Portugal and Spain, with six and 22 officially recognized caprine breeds, encompass 25 % of the European Union goat census. Many of these populations have suffered strong demographic declines because of competition with exotic breeds and the phasing-out of low income rural activities. In this study, we have investigated the consequences of these and other demographic processes on the genetic diversity, population structure and inbreeding levels of Iberian and Atlantic goats.A sample of 975 individuals representing 25 officially recognized breeds from Portugal and Spain, two small populations not officially recognized (Formentera and Ajuí goats) and two ecotypes of the Tinerfeña and Blanca Celtibérica breeds were genotyped with a panel of 20 microsatellite markers. A wide array of population genetics methods was applied to make inferences about the genetic relationships and demography of these caprine populations.ST = 0.07; P < 0.001), which is probably the consequence of their short splitting times and extensive gene flow due to transhumance. In contrast, Canarian goats were strongly differentiated because of prolonged geographic isolation. Most populations displayed considerable levels of diversity (mean He = 0.65).Genetic differentiation among Portuguese and Spanish breeds was weak but significant  contains supplementary material, which is available to authorized users. Goat production is a major economic activity in the Mediterranean basin, with key cultural and environmental implications, especially for smallholders and producers in marginal regions. Many local breeds that are well adapted to harsh conditions have been developed throughout the centuries in order to produce milk, meat, leather and fiber products. However, intensification of production systems since the second part of the 20th century has resulted in the demographic regression of many local populations and the concurrent expansion of a few high-producing exotic breeds . IntensiThe importance of goat production in the Iberian Peninsula is well illustrated by the fact that it holds nearly 25 % of the caprine census of the European Union . There aA total of 975 individuals from 29 local populations in Portugal and Spain were sampled for this study Table ; Fig. 1.BM1329, BM6506, BM6526, BM8125, CRSM60, CSRD247, ETH010, ETH225, HAUT27, ILSTS011, INRA063, MAF065, MAF209, McM527, MM12, OarFCB048, OarFCB304, SPS115, SRCRSP08, and TGLA122  according to the methodology described by Walsh et al. . Twenty o) and unbiased expected (He) heterozygosities, and mean number of alleles (MNA) per population were calculated using the MICROSATELLITE TOOLKIT software [FST distances derived from the 29 goat populations studied was generated with R-lequin [FST and their 95 % confidence interval across loci after 1000 bootstraps were calculated using the GENETIX 4.05 software [FSTP values after 1000 permutations of individuals across the entire population. A Mantel test [FIS was examined [FIS and missing data for a given locus indicates that amplification failure is due to individuals that carry a null allele in the homozygous state.Allele frequencies for each locus, total number of alleles per locus (NA), observed (Hsoftware . Weir ansoftware  F-statissoftware . A graphR-lequin . The measoftware . The samtel test  was perftel test  to test tel test . The relexamined  with theexamined . A positq) in a given ancestral population, as obtained with STRUCTURE, were visualized using DISTRUCT [k)2, where qk is the average fraction of the genetic ancestry of a given breed that belongs to the kth ancestral population, estimated by STRUCTURE analyses [Q) were converted into genetic distances among breeds, following the methodology described by Cañón et al. [The model-based clustering program STRUCTURE  was usedDISTRUCT . The deganalyses . The corn et al. . In thisn et al.  was consn et al. . Informan et al. , 28. Then et al.  and the n et al.  was usedGenetic structure was further investigated by a factorial analysis of correspondence (FAC) using the function “AFC 3D by populations” implemented in GENETIX 4.05 , which iMAF209) to 22 (OarFCB304), with an average of 11.8  and observed (Ho) heterozygosities per locus across all breeds ranged from 0.199 (ETH225) to 0.86 (MM12), and from 0.20 (ETH225) to 0.80 (MM12), respectively. The overall means across loci and breeds for He and Ho were equal to 0.65 and 0.61, respectively  are in Table e per breed ranged from 0.50  to 0.70 (Florida and Pirenaica). Of the 29 populations studied, 12 exhibited a highly significant deficiency in heterozygotes (P < 0.001) and, in this regard, the Verata (0.18) and Preta de Montesinho (0.16) breeds showed the highest within-breed FIS coefficients (Table OarFCB304 and SPS115) did not adjust to the HW equilibrium model for more than one breed (P < 0.001), as shown in Additional file The 20 microsatellite markers analyzed were highly variable, with a total of 236 alleles across all populations. The number of alleles per locus ranged from 3 (SPS115 (9.7 %) and INRA063 (6.6 %) .Frequencies of null alleles ranged from 1.2 % for Tenerife Sur to 7.7 % for Verata breeds , while the amount of differentiation among populations  was significant (P < 0.001), and the inbreeding coefficient of individuals relative to the total population (FIT) was equal to 0.13 . The assessment of genetic distances among breeds based on FST values  between FST genetic distances and geographic distances among breed pairs , although this value dropped to 0.029 when Canarian breeds were removed from the analysis  for K = 3  was estimated between breed HThe genetic structure of the 29 Portuguese and Spanish breeds was further assessed with FAC clustering methods. When all breeds were included in the analysis, the first two axes contributed 27.4 and 8.1 % of the total inertia, respectively Fig. a. The CaWe studied 29 autochthonous goat populations that were sampled in Portugal and Spain by using 20 microsatellite markers in a survey that included endangered populations , commercial goat breeds with larger census  and a feral population from the Canary Islands (Ajuí). Estimates of genetic diversity and analyses of population structure with microsatellites were previously reported for certain specific Iberian goat populations , 7, 30 bIn several instances, the between-breed genetic affinities reported in our study were quite unexpected. For example, the Payoya breed (from the southern Mediterranean coast of the Peninsula) clustered with the Formentera population  in the absence of any historical record that would justify such a relationship. However, an in-depth analysis of this cluster allowed for a clear separation of the two breeds, thus suggesting that migration to the Balearic Islands may have occurred in the past. A close relationship was expected to exist between the Moncaína and Guadarrama breeds, given their very close geographic distribution and common origin, and the fact that these two populations are officially considered as varieties of the Pirenaica breed. However, the analysis with STRUCTURE revealed a close relationship between Pirenaica and Moncaína goats, while the Guadarrama breed was clearly separated from both populations.We were unable to detect a clear differentiation between several of the breeds analyzed Figs. , 5, an oThe Canarian breeds showed a strong genetic differentiation from their Iberian and Balearic counterparts, probably because they descend from the North African goat populations that were introduced by the first Imazighen settlers of the Canary Islands , 39. AftFIS estimate obtained for this breed  as well as other factors . AI is oe of 0.65 for all microsatellite loci analyzed. At the breed level, the mean number of alleles per locus was about 6, with an allelic richness corrected for sample size of about 5 and a He of about 0.65. These values are greater than those found for Asian goats [Overall, we found that genetic diversity was high within most goat breeds studied here, with an overall mean of ~12 alleles/locus and an average Han goats , and siman goats , 43–46. FIS values, which indicates a deficiency in heterozygotes that could be due to inbreeding, the Wahlund effect (population substructure), and other causes [FIS values, but at present it is difficult to infer if this observation is due to population structure, inbreeding or a combination of both factors. As previously said, in the Pitiüsa population, the census is rather small, so inbreeding may have contributed significantly to the high FIS that we detected (Table FIS = 0.183), there are over 8000 breeding females and the rather high FIS observed may reflect the existence of population substructure.Among the Portuguese and Spanish goat breeds, 12 out of 29 showed significantly positive r causes . The VerOverall, we found that the levels of diversity were high, although this finding should not give rise to an excessive optimism because many of the breeds that we analyzed are undergoing a steady and sustained demographic decline that may lead, in the worst case scenario, to their disappearance. One of the most compelling cases is the Blanca de Rasquera population, which in the first half of the 20th century had a census of 30,000 individuals , whereasFIS values, which could be due to a variety of reasons, such as inbreeding and the Wahlund effect. Although, overall, the levels of diversity that we observed are high, these results should be taken with caution, because many of the breeds analyzed are undergoing a steady and sustained demographic decline that could lead to their disappearance. Management and breeding programs based on genetic data should be undertaken in order to minimize inbreeding, maintain overall genetic and allelic diversities and breed identities, and also account for within-breed genetic structure.Our results indicate that the levels of genetic diversity are high in the native goat breeds from Portugal and Spain. Indeed, local breeds, often with a small census, are important reservoirs of genetic diversity. With regard to population structure, Portuguese and Spanish breeds were weakly differentiated. In contrast, Canarian breeds showed a strong genetic differentiation from their Iberian and Balearic counterparts, probably due to influences from their North African counterparts combined with continued geographic isolation. Among the Portuguese and Spanish populations, 12 out of 29 showed significantly positive


The Kalash represent an enigmatic isolated population of Indo-European speakers who have been living for centuries in the Hindu Kush mountain ranges of present-day Pakistan. Previous Y chromosome and mitochondrial DNA markers provided no support for their claimed Greek descent following Alexander III of Macedon's invasion of this region, and analysis of autosomal loci provided evidence of a strong genetic bottleneck. To understand their origins and demography further, we genotyped 23 unrelated Kalash samples on the Illumina HumanOmni2.5M-8 BeadChip and sequenced one male individual at high coverage on an Illumina HiSeq 2000. Comparison with published data from ancient hunter-gatherers and European farmers showed that the Kalash share genetic drift with the Paleolithic Siberian hunter-gatherers and might represent an extremely drifted ancient northern Eurasian population that also contributed to European and Near Eastern ancestry. Since the split from other South Asian populations, the Kalash have maintained a low long-term effective population size  and experienced no detectable gene flow from their geographic neighbors in Pakistan or from other extant Eurasian populations. The mean time of divergence between the Kalash and other populations currently residing in this region was estimated to be 11,800  years ago, and thus they represent present-day descendants of some of the earliest migrants into the Indian sub-continent from West Asia. Human populations show subtle allele-frequency differences that lead to geographical structure, and available methods thus allow individuals to be clustered according to genetic information into groups that correspond to geographical regions. In an early worldwide survey of this kind, division into five clusters unsurprisingly identified (1) Africans, (2) a widespread group including Europeans, Middle Easterners, and South Asians, (3) East Asians, (4) Oceanians, and (5) Native Americans. However, division into six groups led to a more surprising finding: the sixth group consisted of a single population, the Kalash.To further investigate the Kalash population’s demographic history and origins, we genotyped additional unrelated Kalash samples on the Illumina bead chip and sequenced one male individual at high coverage. Our aim was to assess whether the Kalash were a recent or an ancient isolate and categorize the extent of genetic isolation and admixture, if any, with extant or archaic humans and thus better understand the reasons for their unique position in worldwide comparisons.The Kalash samples were collected from three valleys in the Hindu Kush mountain ranges in northwest Pakistan A. In accHigh-coverage (30×) 100-bp paired-end sequencing of one of the genotyped male samples was carried out on an Illumina HiSeq 2000 with 5 μg of lymphoblastoid cell line DNA, standard library preparation, and analysis pipelines developed for the 1000 Genomes Project.2 > 0.4) linkage disequilibrium (LD) and individuals with high identity by descent (IBD) (PLINK IBD score > 0.6) from the analysis. The high IBD threshold was chosen to account for the increased inbreeding levels introduced by the strong genetic drift experienced by the Kalash population. Principal-component analysis (PCA) was performed with EIGENSOFT v.5.01, and ancestry analysis was performed with ADMIXTURE v.1.22.f3) statistics,The data were merged with reference-population data from African and non-African sources covering Eurasia .5,10–12,We applied pairwise sequentially Markovian coalescent (PSMC) analysis to draw inferences about the long-term effective population sizes and times of divergence from ten high-coverage genomes, including the Kalash.f3 statistics.f3  is a function of the shared genetic history of A and B. We used the YRI as an outgroup to non-African populations and computed f3 statistic  to investigate the shared history of an ancient genome and a set of 32 worldwide populations, including the Kalash (X), and f3 statistic  to investigate the Kalash and a set of 32 worldwide populations and ancient genomes (Y). Ancient genomes used in the analysis included the Mal’ta boy (MA-1), a Paleolithic Siberian hunter-gatherer;We assessed the genetic relatedness of ancient genomes to modern populations by computing outgroup ST values and corrected for the effective population size of the population considered (Nep), as shown inSelection in the Kalash populations was estimated as described by Yi et al.KC), between the Kalash and Balochi (TKB)—who were considered representative of the other Pakistani populations—and between the CHB and Balochi (TCB). The population branch statistic (PBS), which defines the length of the branch leading to the Kalash since the split from East Asians, is equal toFor each marker, we calculated T between the Kalash and CHB  = 2,319–2,603]) with the NeON R package. The variants within the 99th percentile of the distribution of our PBS values were annotated, and a list of genes associated with these variants was used for Ingenuity Pathway Analysis (IPA).NWe modeled the effect of drift and selection on specific variants by using the simuPOP library.PCA using only Eurasian and South Asian populations separated the populations from Europe, Caucasus, and West Asia from East Asians in the first component and from South and Central Asians in the second component; Central Asians lay closer to the Sherpa from Nepal and CHB from East Asia. The Kalash samples clustered together as an outlier population to the other South Asian samples from India and Pakistan B. The Kaf3 test statistic  than that of neighboring populations from Pakistan , and six non-African genomes showed that the Kalash, like other non-Africans, experienced a severe bottleneck 50,000–70,000 years ago. The Kalash recovered slightly after the bottleneck but never achieved an effective population size above 20,000, as observed in the GIH (the other South Asian genome) and other non-African genomes, except the MXL A. The KaPakistan B, althouTo examine the time of divergence between the Kalash and other genomes, we used multiple sequentially Markovian coalescent (MSMC) analysis on phased high-coverage genomes. The estimates based on pairs of genomes showed that the Kalash split first from Africans  and then from East Asians (CHB and JPT). The split from Europeans (CEU and TSI) and South Asians (GIH) appears to have happened around the same time C, approxf3 statistics. The measure circumvents potential bias from classical genetic relatedness tests, such as PCA (which has sample-size bias) and FST (which is sensitive to genetic drift that has occurred since divergence of the test populations), when using ancient genomes. According to outgroup f3 statistics, the Kalash share a high level of genetic drift with MA-1, a Paleolithic Siberian hunter-gatherer skeleton dated to ∼24,000 years ago, but not a very high level with La Braña 1, the Mesolithic European hunter-gatherer  or the European farmers represented by Ötzi, the Tyrolean Iceman dated to ∼5,300 years ago  and ACTN3 (MIM: 102574) (−9).We also examined the effect of the inferred long-term isolation on genetic drift and natural selection in the Kalash. Our PBS analysis showed evidence of possible positive selection on 1,709 SNPs, of which 762 lie within 548 genes, including  102574) . IPA shoLCT [MIM: 603202]) expression and confers lactose tolerance, is fixed for the ancestral lactose-intolerant allele in the Kalash. The derived allele, however, is reported to be present at a moderate frequency (average 29%) in Pakistan.Two variants that were highly differentiated between the Kalash and the neighboring Pakistani populations stood out. One variant, rs4988235 (c.−13910C>T), which influences lactase , is a natural knockout variant in f3, and linkage-based statistics. The fact that researchers also genotyped ten of these samples earlier by using the HGDP-CEPH panel and that these cluster with the samples genotyped in this study rules out the possibility of confounding results due to population sub-structure within the Kalash.The present study sheds light on the origins of the enigmatic Kalash population from Pakistan. We propose that the population represents an ancient genetic isolate rather than a recently split population showing extreme genetic drift, as suggested by earlier studies.The ancient separation of the Kalash from a common Eurasian ancestor is supported by PSMC and MSMC analyses, which estimated that the Kalash split from East Asians (CHB and JPT as proxy) prior to splitting from Europeans and other South Asian populations. The split from Europeans (CEU and TSI) and South Asians (represented here by GIH) appears to have occurred during the Neolithic period, which is also supported by the decay of LD. LD decay showed that the Kalash were the first population to split from the other Central and South Asian cluster around 11,800  years ago. This estimate remained constant even after the addition of an African (YRI) population or when the Kalash were compared with different subsets of non-African populations. The pairwise times of divergence with other Pakistani populations ranged from 8,800 years ago with the Burusho to 12,200 years ago with the Hazara. Although migration and undetected admixture in reference populations could bias our estimate of the time of divergence, using different subsets of population revealed no strong bias in the split between the Kalash and South Asians, which occurred after the split between Europeans and South Asian populations.e of around 2,500 , estimated from LD decay with no evidence of admixture. These Ne estimates are lower than those obtained from PSMC analysis because the latter method gives a single estimate of the cross-coalescence rate from the present to 24,000 years ago, whereas the linkage-based method gives us several estimates over the past 10,000 years. It is likely that PSMC analysis could not detect that the Kalash population suffered a continuous decline in effective population size. Taking into account the expected differences in Ne between autosomes and the Y chromosome, this is in agreement with the reported Ne of 237–1,124, which was estimated with observed and evolutionary mutation rates for Y chromosomal STRs.Since this split, the Kalash have maintained a low NThe Kalash represent a unique branch in the South Asian population tree and appear to be the earliest population to split from the ancestral Pakistani and Indian populations, indicating a complex scenario for population origins in the sub-continent rather than just the ancestral northern and southern Indian components identified previously.Whereas the Kalash have recently been reported to have European admixture, postulated to be related to Alexander’s invasion of South Asia,We observed that the Kalash share a substantial proportion of drift with a Paleolithic ancient Siberian hunter-gatherer, who has been suggested to represent a third northern Eurasian genetic ancestry component for present-day Europeans.LCT-MCM6 (MIM: 601806) region in this population.We also looked at how this long-term separation, isolation, and low effective population size affected the patterns of genetic variation in the Kalash. One striking example is the frequency of the derived allele for rs4988235, which has been linked to lactose tolerance. The Kalash, like the MA-1, are fixed for the ancestral allele for this variant, whereas their neighbors in Pakistan have been observed to have moderate frequencies of the derived allele. Although this supports their long-term isolation, it is surprising in other ways because the Kalash have no reported lactose intolerance and indeed celebrate a “milk day” during their annual spring rituals.ACTN3 variant (rs1815739) associated with normal variation in human muscle strength and speed.ACTN3 has not been associated with adaptation to high altitude, RYR2, another gene with an intronic outlier variant (rs2992644) in PBS, has.Another example is the extremely high frequency (93%) of the stop-gain It has been postulated that South Asia, which is now a densely occupied land, was encountered by the first populations of modern humans that ventured out of Africa more than 50,000 years ago. The exact route taken by these earliest settlers is not known, although it has been suggested that they traveled via a southern coastal route.


Cycas species with resembling morphology and overlapped distribution range along the Red River (Yuanjiang) in China: Cycas diananensis Z. T. Guan et G. D. Tao, Cycas parvula S. L. Yang and Cycas multiovula D. Y. Wang. A total of 137 individuals from 15 populations were genotyped by using three chloroplastic  and two single copy nuclear (RPB1 and SmHP) DNA sequences. Basing on the carefully morphological comparison and cladistic haplotype aggregation (CHA) analysis, we propose all the populations as one species, with the rest two incorporated into C. diannanensis. Genetic diversity and structure analysis of the conflated C. diannanensis revealed this species possessed a relative lower genetic diversity than estimates of other Cycas species. The higher genetic diversity among populations and relative lower genetic diversity within populations, as well as obvious genetic differentiation among populations inferred from chloroplastic DNA (cpDNA) suggested a recent genetic loss within this protected species. Additionally, a clear genetic structure of C. diannanensis corresponding with geography was detected based on cpDNA, dividing its population ranges into “Yuanjiang-Nanhun” basin and “Ejia-Jiepai” basin groups. Demographical history analyses based on combined cpDNA and one nuclear DNA (nDNA) SmHP both showed the population size of C. diannanensis began to decrease in Quaternary glaciation with no subsequent expansion, while another nDNA RPB1 revealed a more recent sudden expansion after long-term population size contraction, suggesting its probable bottleneck events in history. Our findings offer grounded views for clarifying species boundaries of C. diannanensis when determining the conservation objectives. For operational guidelines, the downstream populations which occupy high and peculiar haplotypes should be given prior in-situ conservation. In addition, ex-situ conservation and reintroduction measures for decades of generations are supplemented for improving the population size and genetic diversity of the endemic and endangered species.Delimitating species boundaries could be of critical importance when evaluating the species' evolving process and providing guidelines for conservation genetics. Here, species delimitation was carried out on three endemic and endangered  Cycas L. (Cycadaceae), which is considered as the basal lineage of the Cycadales, and also the sister group to the other gymnosperms  that is resulted from the uplifting of Himalaya and the basin expansion of South China Sea . Among them, materials of C. parvula and C. multiovula were obtained from cultivated individuals in the village after the verification that they were from the same population. The C. diannanensis population ZSM and C. multiovula population ZSD were sampled from the same place . Information of sampling sites and the number of individuals from each population used in this study are presented in Table A total of 137 individuals were selected for subsequent analysis from the 15 populations collected along the Red River region in China, including 10 populations of psbA-trnH, rps4-trnL, and atpI-atpH  for complete analysis , 2.0 μL dNTPs (10 mM), 4.0 μL 10 × PCR buffer, 0.6 μL of each primer, 0.6 μL Taq DNA polymerase (5 U/μL)  and 26 μL double-distilled water. For nDNA, the PCR reactions contained 40 ng DNA, 2.4 μL MgCl2 (25 mM), 2.0 μL dNTPs (10 mM), 2.0 μL DMSO, 4.0 μL 10 × PCR buffer, 0.7 μL of each primer, 0.7 μL Taq DNA polymerase (5 U/μL)  and 24.6 μL double-distilled water. PCR amplifications were performed in a thermocycler under the following conditions: an initial 5 min denaturation at 80°C, followed by 34 cycles of 1 min at 95°C, 1 min annealing at 50°C, and a 1.5 min extension at 65°C, and a final extension for 10 min at 65°C for cpDNA intergenic spacers. For nDNA sequences, an procedure of initial 4 min denaturation at 94°C, which was followed by 34 cycles of 50 s at 94°C, 1 min annealing at 50°C (for SmHP) or 55°C (for RPB1), and a 1.5 min extension at 72°C, and a final extension for 10 min at 72°C was used. All PCR products were sequenced in both directions with the same primers for the amplification reactions, using an ABI 3770 automated sequencer at Shanghai Sangon Biological Engineering Technology & Services Company Ltd. The individuals with nDNA sequences which had one or more heterozygous sites in the first sequencing round were subsequently cloned. PCR products were purified using the TIANgel Midi Purification Kit (Tiangen). Purified products were linked to pMD18-T Vector and then inserted to E. coli DH5α strains. Six to ten clones were randomly picked and sequenced until the heterozygous site split into two alleles. The data sets of the DNA sequencing in this study were deposited in GenBank (accession numbers from KT334601–KT334653).Materials for DNA extraction were from young and healthy leaves which were collected and dried immediately in silica gel. Genomic DNA was extracted from dried leaves using the modified CTAB method Doyle, . ApproxiP = 0.4, < 0.5) by PAUP* 4.0b10  indices through DnaSP software as well. The within-population gene diversity (HS), gene diversity in total populations (HT) and two coefficient of population differentiation, GST and NST were calculated by Permut v1.0 (http://www.pierroton.inra.fr/genetics/labo/Software/Permut).Haplotypes from five markers for all the three species were calculated from aligned DNA sequences by DnaSP v5.0   mutation per site per year for cpDNA and nDNA respectively, which had previously been estimated in seed plants for synonymous sites  values in three pre-runs. The ESS parameter was expected to surpass 200, which suggested acceptable mixing and sufficient sampling in analysis. Adequate sampling and convergence to the stationary distribution were checked using Tracer v1.6  sequence matrix was aligned with a consensus length of 664bp, which contained 15 substitution sites, and formed 12 nuclear haplotypes in the 15 populations. The most widely distributed haplotypes were Hap S2 and S3, which occurred in 12 and 10 populations respectively and were shared by all the three species  sequence matrix was aligned with an accordant length of 912bp, in which six substitutions were detected, deriving 11 nuclear haplotypes in total  analysis and Bayesian inference of cpDNA and nDNA haplotypes generated similar cladograms corresponding to the network analysis, whereas differed in the support values located on internal nodes. For cpDNA, all 13 haplotypes appeared as a distinct comb-like structure with three paraphyletic subclades nested inside. Within the cladogram, haplotypes H12, H11, and H3 clustered in the same subclade which was occupied by e Figure . As the SmHP, the first divergent haplotypes were Hap S1, S3, and S8, which were widely shared by all the populations  which was mapped in middle of the network evolution diagram. The second most frequent haplotype Hap S3 located at the top of the haplotype cladogram and the near margin of network evolution diagram, suggesting it a recent evolved haplotype. Similarly, haplotypes aggregation of RPB1 in each branch (clade) from the cladogram neither matched with the species populations nor geographical distributions , SmHP: 2.048 Myr, RPB1: 2.571 Myr), indicating a haplotype splitting in late Pliocene  or within Pleistocene (2.6 Myr). Estimate time of different haplotypes on the internal divergence node was displayed on Figure Inference of divergent time of haplotypes from our cpDNA and nDNA data all suggested a recent divergence of the SmHP and RPB1) showed well supported lineage clades (most PP > 90) of the three species  diversity in all populations were detected in combined cpDNA  than in nDNA  was higher than the average intra-population diversity . For cpDNA, most populations displayed no haplotype diversity except population GJD, MHG, HTP, YJH, and YYM, which distributed along downstream of the Red River. However, most populations occupied high haplotype diversities at the nDNA level.Relative lower total nucleotide (Pi) and haplotype  of isolation by distance (IBD) by combined cpDNA data, indicating a positive correlation between genetic and geographical distance, while both two nDNA markers showed non-significant  correlations between such distances  as well as one C. diannanensis population (MHG). The results of two nDNA sequences also both suggested K = 2 , indicating these populations had not undergone a recent population expansion. While result inferred from nuclear RPB1 gene showed a unimodal curve, suggesting that population had experienced bottleneck events in history.Meanwhile, the results of the mismatch analysis Figure  for all SmHP) of the investigated populations during Quaternary glaciations. While for nDNA RPB1, a quite recent subsequent expansion after historical population decreasing  : 1-2.C. pectinata var. manhaoensis C. J. Chen & P. Yun, Acta Bot. Yunnan. 17(4): 400, 1995; C. parvula S. L. Yang & D. Y. Wang, Cycads in China. 93. 1996; C. multiovula D. Y. Wang, Cycads in China. 83. 1996.C. diannanensis reveals it possesses higher haplotype diversity and total genetic diversity  than C. debaoensis        when compared with C. simplicipinna     and nDNA . Particularly, a distinct phylogeographical structure with cpDNA haplotypes distribution was revealed by the result of U test  of three markers in this study, a possible similar population contraction may appear in C. diannanensis in history from the results of Bayesian skyline plots  by Bayesian skyline and the possible bottleneck events deducted from mismatch analysis. This discordance may be attributed to historical genetic drift or larger selective pressure existed on this gene  into several isolated surviving populations during the glaciation, with relative lower genetic diversity being detected in this study.Glaciations, especially Pleistocene glaciations made deep effects on the spatial distribution of plants Hewitt, . These s Figures . Mismatc Figures  but a po Figures , 7C. Mea Figures , we arguCycas species distributed in southwest China had all experienced population retreats . The fault region harbors numerous of Cycas species in its ranges, especially in the dry hot valley of southwest China  are conserved by cultivation in local regions, seed or seedling reproduction from the two populations are suggested to artificially introduce the genetic components into other regions or in the wild to increase the individual number and genetic diversity of each population.Cycads, for their palm-like leaves and abundant symbols in tradition, are often cultivated as ornaments or traded for medical value (Cousins et al., KT334601-KT334653.The data set of the DNA sequencing data in our study are deposited in GenBank under accession numbers The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Scinax eurydice, testing the congruence among patterns identified and proposed by the literature for Pleistocene refugia, microrefugia, and geographic barriers to gene flow such as major rivers. Furthermore, we aim to evaluate predictions of such barriers and refugia on the genetic structure of the species, such as presence/absence of dispersal, timing since separation, and population expansions/contractions. We sequenced mitochondrial and nuclear genetic markers on 94 tissue samples from 41 localities. We inferred a gene tree and estimated genetic distances using mtDNA sequences. We then ran population clustering and assignment methods, AMOVA, and estimated migration rates among populations identified through mtDNA and nDNA analyses. We used a dated species tree, skyline plots, and summary statistics to evaluate concordance between population’s distributions and geographic barriers and Pleistocene refugia. Scinax eurydice showed high mtDNA divergences and four clearly distinct mtDNA lineages. Species tree and population assignment tests supported the existence of two major clades corresponding to northeastern and southeastern Atlantic Forest in Brazil, each one composed of two other clades. Lineage splitting events occurred from late Pliocene to Pleistocene. We identified demographic expansions in two clades, and inexistent to low levels of migrations among different populations. Genetic patterns and demographic data support the existence of two northern Refuge and corroborate microrefugia south of the Doce/Jequitinhonha Rivers biogeographic divide. The results agree with a scenario of recent demographic expansion of lowland taxa. Scinax eurydice comprises a species complex, harboring undescribed taxa consistent with Pleistocene refugia. Two rivers lie at the boundaries among populations and endorse their role as secondary barriers to gene flow.We aim to evaluate the genetic structure of an Atlantic Forest amphibian species,  The Atlantic Forest (hereafter AF) harbors one of the richest, most threatened and unique biotas in the world . A high Biodiversity is not evenly distributed in the AF, with centers of endemism supported by faunal inventories, phylogenetic analyses and phylogeographic studies ,11. PhylGeographic barriers associated to neotectonic activity have been evoked to explain evolution of biota from the AF . BiogeogPleistocene climatic fluctuations played a central role in the stability, biodiversity distribution, and phylogeographic endemism in the Brazilian Atlantic Forest –5. ForesScinax eurydice is widely distributed in the Atlantic Forest and is a species that occupies the forest edge, being able to use temporary and/or permanent pounds on the outer margin and adjacent open areas in the forest for breeding  recovered 4 as the best number of k : (1) variation among groups = 89% and variation within groups = 6% for ND2; (2) variation among groups = 31% and variation within groups = 13% for RAG1; (3) variation among groups = 40% and variation within groups = 16% for β-fibint7 and (4) variation among groups = 82% and variation within groups = 17% for 28S .We found moderate  to low  within-clade genetic divergences. Mitochondrial haplotype diversity ranged from 95% (± 9.6%) in NE1 to 99% (± 12.6) in SE1. Nucleotide diversity values for SE1 and SE2 were higher than NE1 and NE2 . 28S shoThe *BEAST species tree based on the combined dataset (mtDNA and nDNA) was similar to the mitochondrial phylogeny. The basal split  and the relationships among northern  and southern populations  were all strongly supported .The dated chronogram generated in *BEAST revealed a recent history of diversification, with lineage splitting events occurring from late Pliocene to Pleistocene [3.9–0.4 Million of years (Myr); Mean values of migration rates for two-population and four-population models are largely in congruent. However, peak estimates for migration from SE2 to SE1 and from NE2 to SE1 are much lower in the four-population model  Figs. OnNeutrality and population size change tests for ND2 indicated significant signals of demographic expansion in clades NE2 and SE2 . For RAG1 D and Fs tests were significant for samples comprising clade NE2 and for β-fibint7 only the Fs test was significant for samples that correspond to clade SE2. We did not find any evidence for demographic expansion in 28S . These rS. eurydice  experienced distinct demographic histories. Changes in effective population sizes (Ne) through time were observed only for SE1 and NE2. We evidenced a reduction of Ne in clade SE1, which started about 130,000 years ago  and between northern and southern clades (Jequitinhonha River). Nevertheless, the signal for these barriers is not pervasive for all markers, and no river can be pointed as a barrier between the two southern mitochondrial clades.Although not necessarily being the sole cause of such breaks, rivers can act as secondary barriers, partially restricting gene flow among populations . This seIndeed, nuclear and mitochondrial markers frequently show different phylogeographic resolutions for the same biogeographic history due to differences in mutation rates, recombination, and effective population sizes ,56. Suchtepuis of Venezuela, and their biogeographic significance for the establishment of present day biotas is undisputable [Microrefugia have been originally proposed to help explain the persistence in time of high-mountain species and communities in the sputable . In the sputable . Althougsputable , they alsputable ,21,58,59sputable . In factsputable ,60.S. eurydice exhibit spatial and temporal concordance with previous works, which recovered a range of temporal divergences between the Pliocene and Pleistocene in the AF [S. eurydice is consistent with allopatric diversification associated with Pliocene-Pleistocene climatic cycles. The two divergent clades in the Bahia and Pernambuco refuges that do not exchange migrants, the significant migration from NE to SE, and a highly divergent mitochondrial clade in high altitude areas at the southern portion of the AF support both refuges predicted north of the Doce River [S. eurydice may have had its distribution fragmented and restricted to microrefuges in the southern AF and to large refuges in Pernambuco and Bahia, with a recent re-establishment of contact by a directional migration from climatically-stable northeastern populations to the southeast and from lowland southeastern populations (SE2) to upland populations (SE1).Pleistocene climatic fluctuations have been implicated in the diversification of several animal taxa in the AF ,16–19,61n the AF ,52. Phylce River –5 and thce River ,5,13. HeS. eurydice occupy less stable portions of the AF [S. eurydice may actually represent more than one species. Future researches with S. eurydice populations using an integrative approach and inferences of hybridization in secondary contact zones could be used to describe and delimit the spatial patterns of variation in their geographic range in Atlantic Forest and provide a more accurate hypothesis for its taxonomy.During the Last Glacial Maximum rainforests were restricted to mountainous areas and high altitude species were less affected by cold and dry climates of Pleistocene glaciations . Lowlandf the AF , they shf the AF  and birdf the AF  suggestiScinax eurydice is genetically structured across the Atlantic Forest, showing high mtDNA divergence and low to inexistent levels of migration among populations. Genetic patterns and demographic data match predictions for the existence of large Refugia in the Northern AF (Bahia and Pernambuco), as well as microrefugia south of the Doce/Jequitinhonha Rivers biogeographic division. Patterns of gene flow, demography, and divergence dates agree with a scenario of recent demographic expansion of lowland taxa, likely favored by the end of major glaciation events and the expansion of suitable habitats. Species tree, demographic parameters, temporal and spatial population structures recovered endorse S. eurydice as a complex of species. Two rivers lie at the boundaries among populations (Jequitinhonha and São Francisco), a pattern repeatedly found in the AF, which suggests that the role of rivers as secondary barriers must be re-evaluated. The current distribution of these populations and their origin are linked to the recent history of the AF, underscoring the importance of taxonomic reappraisals based on multiple lines of evidences to ensure the preservation not only of species, but of the evolutionary processes responsible for the origin of current biodiversity.S1 Fig(PDF)Click here for additional data file.S2 Fig−7).(A) Divergence estimates of the six two-population models. Each curve represents the estimated divergence time between two populations. (B) Divergence time estimates resulting from a four-population model. Each curve represents one of the three divergence parameters of a four-population model. Transformations were performed using the geometric mean of the mutation rates (7.52 x 10(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig−7).Transformations were performed using the geometric mean of the mutation rates (7.52 x 10(PDF)Click here for additional data file.S6 Fig−7).Transformations were performed using the geometric mean of the mutation rates (7.52 x 10(PDF)Click here for additional data file.S1 TableAbbreviations of Brazilian states: PB, Paraíba; PE, Pernambuco; AL, Alagoas; SE, Sergipe; BA, Bahia; ES, Espírito Santo; MG, Minas Gerais; RJ, Rio de Janeiro; SP, São Paulo. Collection acronyms: CHUFPB, Coleção Herpetológica da Universidade Federal da Paraíba; URCA-H, Coleção Herpetológica da Universidade Regional do Cariri; UFBA, Museu de Zoologia da Universidade Federal da Bahia; CFBH; Coleção Célio F.B. Haddad, Departamento de Zoologia, Universidade Estadual Paulista “Júlio de Mesquita Filho”; MNRJ, Museu Nacional, Rio de Janeiro; JC-MTR, LSH-MTR and MTR, Coleção Miguel Trefaut Rodrigues, deposited at the Universidade de São Paulo; PUCMG, Coleção de Herpetologia do Museu de Ciências Naturais da Pontifícia Universidade Católica de Minas; UFMG; Coleção de Herpetologia da Universidade Federal de Minas Gerais; MZUFV, Museu de Zoologia João Moojen, Universidade Federal de Viçosa; IIBP-H; Colección Herpetológica del Instituto de Investigación Biológica del Paraguay, Asunción, Paraguay.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.


Lampornis amethystinus is distributed in northern Mesoamerica, with geographically disjunct populations. Based on sampling across the species range using mitochondrial DNA (mtDNA) sequences and nuclear microsatellites jointly analysed with phenotypic and climatic data, we (1) test whether the fragmented distribution is correlated with main evolutionary lineages, (2) assess body size and plumage color differentiation of populations in geographic isolation, and (3) evaluate a set of divergence scenarios and demographic patterns of the hummingbird populations. Analysis of genetic variation revealed four main groups: blue‐throated populations (Sierra Madre del Sur); two groups of amethyst‐throated populations ; and populations east of the Isthmus of Tehuantepec (IT) with males showing an amethyst throat. The most basal split is estimated to have originated in the Pleistocene, 2.39–0.57 million years ago (MYA), and corresponded to groups of populations separated by the IT. However, the estimated recent divergence time between blue‐ and amethyst‐throated populations does not correspond to the 2‐MY needed to be in isolation for substantial plumage divergence, likely because structurally iridescent colors are more malleable than others. Results of species distribution modeling and Approximate Bayesian Computation analysis fit a model of lineage divergence west of the Isthmus after the Last Glacial Maximum (LGM), and that the species’ suitable habitat was disjunct during past and current conditions. These results challenge the generality of the contraction/expansion glacial model to cloud forest‐interior species and urges management of cloud forest, a highly vulnerable ecosystem to climate change and currently facing destruction, to prevent further loss of genetic diversity or extinction.The present day distribution and spatial genetic diversity of Mesoamerican biota reflects a long history of responses to habitat change. The hummingbird  Geographic isolation is the most widely accepted mode of speciation by which populations differentiate  (Trochilidae), is examined through phylogeographic and population genetic analyses of nuclear and mitochondrial DNA data. Lampornis amethystinus is a resident hummingbird species to the cloud forest in the Mesoamerican highlands , and margaritae restricted from Michoacán to Oaxaca (possibly sympatric with amethystinus in southern Oaxaca) with gorgets glittering bluish violet , brevirostris (western TMVB), margaritae (Guerrero), circumventus , salvini , and nobilis   found exclusively in the Sierra Madre del Sur evolved recently from other amethyst‐throated lineages distributed in adjacent mountain ranges or instead the blue‐throated form resulted form more complex population divergence scenarios and long‐term isolation. Patterns of morphological, genetic diversity (mtDNA and nuclear microsatellites), haplotype genealogy, and genetic structure were examined to infer the distributional, demographic, and evolutionary history of the amethyst‐throated hummingbird. In addition, we estimated the timing of divergence and gene flow rates between the blue and amethyst lineages to compare the evolution of phenotypic variation with that of major climatic events during the Quaternary. To frame the information derived from genetic analyses in an explicitly paleoecological context, we constructed species distribution models to predict where populations of amethyst‐throated hummingbirds resided during the LGM and Last Interglacial (LIG) climate conditions, and whether populations were connected during those events. The distribution and composition of the Mesoamerican biota have been strongly influenced by geological and climatic events, with considerable invasions of Mesoamerica by South American tropical elements and temperate elements from North America prior to the formation of the Isthmus of Panama ca. 4.5 MYA , we present new sequence data for 88 additional individuals (Table S1). The 157 individuals were sampled from 43 localities and categorized into five groups based on mountain geography: SMO = Sierra Madre Oriental; TUX = Sierra de Los Tuxtlas and Sierra de Santa Marta; SMS = Sierra Madre del Sur ; TMVB = Trans‐Mexican Volcanic Belt; CHIS = Chiapan Highlands separated by the Central Depression that together with Guatemala and El Salvador form the Trans‐Isthmian Highlands region (TIH); Fig. S1 and Table S1). The sampling presented in this study practically covers the entire distribution of amethyst‐throated hummingbirds in Mexico. Birds were captured in mist nets and two rectrices or tissue samples were collected for subsequent genetic analysis. Samples were collected using required permits and approved animal welfare protocols. We also obtained sequence data or downloaded sequences from GenBank for congeners , Lamprolaima rhami, Doricha eliza, Calothorax pulcher, Selasphorus platycercus, and Archilochus colubris to be used as outgroups according to García‐Moreno et al.  and 402 bp of cytochrome b (cyt b) genes were amplified by PCR and sequenced to infer phylogenetic relationships among haplotypes. Genomic DNA was extracted using Chelex (10%) or the extraction DNeasy blood and tissue kit , following the protocol recommended by the manufacturer. Amplification of ND2 was conducted with primers L5215–H5578 , 3.6 mmol/L−1 MgCl2, 0.58 mmol/L−1 dNTPs, 0.4 μg/μL BSA, 0.18 μmol/L−1 of each primer, 0.04 U Taq polymerase (Promega), and 1–1.5 μL of genomic DNA. The PCR cycling conditions for ND2 consisted of an initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 20 sec, annealing at 47°C for 20 sec and an extension of 74°C for 1 min, and a final extension of 72°C for 3 min. For the cyt b the PCR cycling conditions consisted of an initial denaturation at 80°C for 5 min, followed by 35 cycles consisting of denaturation at 95°C for 1 min, and annealing at 47°C for 4 min, and a final extension of 66°C for 10 min. PCR products visualized on 1% agarose gels stained with ethidium bromide were purified with the QIAquick kit  and sequenced using the BigDye Terminator Cycle Sequencing kit . Sequences were read in a 310 automated DNA sequencer  at the INECOL's sequencing facility. Finally, sequences were assembled using Sequencher ver. 5.2.3  and then manually aligned with SE‐AL ver. 2.0a11 (http://evolve.zoo.ox.ac.uk/software.html). All newly acquired sequences have been deposited in GenBank .Two mitochondrial genes—349 base pairs (bp) of NADH nicotinamide dehydrogenase subunit 2  and Selasphorus platycercus . Amplification of microsatellite loci were performed with the Multiplex PCR kit (Qiagen) using two mixes of four fluorescently labelled primers (Applied Biosystems). Multiplex PCR amplification  contained final concentrations of 1× Multiplex PCR Master Mix, 0.2 μmol/L−1 of primer mix, and 0.5 μL of DNA. Alleles visualization and fragment sizing were performed using GENEMAPPER ver. 3.2 (Applied Biosystems) against an internal size standard  and scored manually. A full description of the development protocol for the loci can be found at the Molecular Ecology Resources Database  and nucleotide diversity (π) for each geographical group, and pairwise comparisons of FST values between populations and groups with 1000 permutations were calculated using ARLEQUIN ver. 3.5 (Excoffier and Lischer n = 43), whereas ‘groups’ are sets of pooled populations (n = 5), as specified in Table S1. To determine whether or not populations are geographically structured, three analyses of molecular variance  within populations and loci were calculated using GENEPOP ver. 3.4  was inferred by means of neutrality tests and mismatch distributions constructed in ARLEQUIN. To test whether populations evolved under neutrality, Fu's Fs test and Tajima's D tests were calculated with 1000 permutations, and mismatch distributions were calculated using the sudden expansion model of Schneider and Excoffier (Ne). This analysis was performed for each of the geographic groups separately based on BEAST and AMOVA results with the same settings used in BEAST for divergence time estimation (see below), except that the coalescent tree prior was specified as Bayesian skyline with five groups. Three runs of 10 million steps and effective sample sizes (ESS) > 200 were compared to ensure convergence. Outputs were combined in LOGCOMBINER ver. 1.6.1 (Drummond and Rambaut http://tree.bio.ed.ac.uk/software/tracer/). The time axis was scaled using the geometric mean substitution rate of 0.010075 substitutions per site per lineage per million years (s/s/l/MY), according to the average rates of 0.029 s/s/MY for ND2 and 0.014 s/s/MY for cyt b obtained for Hawaiian honeycreepers  sequences and the closest nucleotide substitution model under the Bayesian information criterion (BIC), GTR, suggested by jMODELTEST ver. 0.1.1 .We estimated divergence times among groups with the Bayesian approach implemented in BEAST using the combined mtDNA  and the two descendant populations (q1 and q2), effective number of migrants per generation in both directions (m1‐to‐2 and m2‐to‐1), and time since divergence (t) at which the ancestral population gave rise to the descendant populations. IM models search parameter space for the most likely estimates using a Bayesian framework assuming random mating within populations and that populations are each other's closest relatives not exchanging genes with other nonsampled populations  coalescent model implemented in IMa (Hey and Nielsen −4 substitutions per site per year (s/s/yr) for the ten loci according to the averages of 2.9 × 10−8 s/s/MY for ND2 and 1.4 × 10−8 s/s/MY for cyt b obtained for Hawaiian honeycreepers  is calculated according to T = a + [s/(1–s)] . To convert time since divergence parameter of IMa to years, t, we divided the time parameter (B) by the mutation rate per year (U) converted to per locus rate by multiplying by the fragment length in base pairs.Initial runs were conducted searching for suitable conditions to constrain parameter intervals and to alter the heating scheme to achieve sufficient mixing among chains (Hey and Nielsen isolation split model 1) predicts that TMVB (Pop1) merged with SMO (Pop2) at t1 then SMO merged with SMS  at t2 and subsequently with CHIS east of IT (Pop4) at t3. This scenario was expected to be the most likely according with hierarchical STRUCTURE and BEAST analyses. The second scenario  is similar to the previous one but predicts that SMS (Pop3) merged with TMVB (Pop1) at t1 then TMVB merged with SMO (Pop2) at t2 and subsequently with CHIS east of IT (Pop4) at t3. The third scenario  consisted of the same basal split between CHIS (Pop4) and the rest of groups west of IT described in previous scenarios but includes a hybridization/lineage fusion event in which SMS (Pop3) is the descendent of admixture between TMVB (Pop1) and SMO (Pop2) at t1, then Pop1 merged with Pop2 at t2, and subsequently with Pop4 at t3. Although there are numerous possible scenarios of divergence, we considered that these three scenarios represent the close relationships among groups and the most likely demographic scenarios during Pleistocene climate cycles  under each scenario to estimate Type I and Type II error rates  and LIG  and whether the conditions for range expansion and population connectivity occurred. We assembled a dataset of occurrences for L. amethystinus from georeferenced museum specimens obtained through http://vertnet.org and the Global Biodiversity Information Facility , supplemented with records from field collection. After careful verification of every data location and removing duplicate occurrence records, we restricted the dataset to unique records for the analyses, leaving 109 unique presence records for L. amethystinus. These localities sample the entire distribution range of each species. Distribution records were input into and analysed with the maximum entropy algorithm in MAXENT ver. 3.2.2  to infer the SDMs. Present‐day temperature and precipitation data (BIO1–BIO19 variables) were drawn as climate layers from the WorldClim database  and 500 iterations, ensuring only one locality per grid cell. We evaluated model performance using cross‐validation running a random dataset using 70% of the occurrence points for training and 30% to test the model and then estimating the area under the receiver operating curve (AUC) of the threshold‐independent receiving operating characteristic curve  and LIG (at 30 arc‐sec), using the x + 1) before statistical analysis.Three body measurements were obtained from each of 242 hummingbirds  from museum specimens  using a We performed a multivariate analysis of variance (MANOVA) followed by one‐way ANOVAs to examine morphological variation among groups of populations with SPSS ver. 17.0 for Mac (SPSS).L. amethystinus females. Feather coloration between the chest and throat of females is dusky cinnamon, a pigment‐based color that is likely constrained in their expression by metabolic pathways  obtained from 85 male museum specimens to which we had permission for plucking see . Bright  = 0.25). We plotted the resulting spectral data to visualize the mean reflectance curves for gorgets from each genetic group (or subspecies). Because we detected two peaks from the spectral curve (see http://www.r-project.org/). Although hues of the second peak are likely outside the range of avian vision, we provide these data to show that there is a previously unnoticed strong signal probably perceived by hummingbirds.The raw data files of measured reflectance spectra of 85 males were first loaded and organized for further analysis in the R package PAVO ver. 0.5–1  for the points in the tetrahedral color space, the angles theta and phi  in radians, which determine the hue of the color, the r vector (r.vec), which measures saturation or the distance from the achromatic centre, the maximum r vector (r.max) achievable for the color's hue, and the r.achieved, which measures the relative r distance from the achromatic centre in relation to the maximum distance achievable (r.vec/r.max). These are receiver‐centric variables that represent reflectance spectra in the avian tetrahedral color space  (Table h) and nucleotide diversity (π) values were high, with samples from CHIS containing the highest levels  followed by those from SMO  respectively  when populations are grouped as separated by the IT .The AMOVA results for mtDNA showed that 61.4% of the genetic variation was explained by differences within populations and 38.6% by differences between populations when all locations were treated as a single group Table . GeneticIT Table . When saIT Table . PairwisAcross groups of populations , the mean number of alleles per locus and allele richness values were moderately high and similar among groups of populations, ranging from 7.4 (SMS) to 5.4 (TMVB) and from 5.4 (CHIS to 4.6 (SMO) respectively (Table S4). Observed heterozygosity values ranging from 0.56 (TMVB) to 0.47 (CHIS) did not consistently deviate from H–W equilibrium (Table S4). The mean expected heterozygosity ranged from 0.76 in the TMVB group to 0.59 in the CHIS group (Table S4). According to MICROCHECKER, two localities departed from H–W equilibrium after Bonferroni corrections at locus CACU16‐1 and HumB15, probably due to the presence of null alleles. No significant linkage disequilibrium was detected in any of the population‐loci comparisons after Bonferroni corrections (Table S5).FST estimate = 0.0635, P < 0.05). Pairwise FST values from comparisons between groups of populations were all statistically significant, with the weakest relationship observed between SMO and SMS (Table S3). The clustering using STRUCTURE not only revealed substantial phylogeographic structure but also recovered hierarchical relationships among groups of populations. Two peaks of ΔK were detected, and the break in the slope of the distribution of L(K) was at K = 2 and K = 4. When K = 2, individuals from the CHIS group were firstly separated from others west of IT  from the SMS geographic group have large assignment Q values (>0.6) indicating their membership in what is otherwise considered the CHIS gene pool , indicating historic demographic expansion events .L. amethystinus samples in a well‐supported monophyletic group . Within this clade, the phylogenetic relationships among haplotypes were split between populations separated by the IT. Individuals from populations east of the Isthmus form two highly supported groups (PP = 0.99), one corresponding to the salvini subspecies south of the Motagua‐Polochic‐Jocotán fault system (MPJ) and the other with samples north of the MPJ fault system in Chiapas. Relationships between samples from the Sierra Madre del Sur (margaritae and circumventus) and those from the Sierra Madre Oriental, Sierra de Los Tuxtlas and the Trans‐Mexican Volcanic Belt (amethystinus and brevirostris) were poorly resolved. Diversification of mtDNA haplotypes in L. amethystinus began in the Pleistocene with the split between IT lineages estimated at 1.49 MYA  and Late Pleistocene ages for the split between CHIS populations separated by the MPJ fault system .The BEAST analysis grouped all IMa results are summarized in Table L. amethystinus. In accordance with the isolation and migration model, ABC estimated a smaller Ne for the descendant populations compared with both N5 and N6 ancestral populations, and a smaller Ne for the TMVB compared with the CHIS, SMS, and SMO populations (Table S6). Estimated mean mutation rates of mtDNA and microsatellites were estimated to be 0.000187 and 0.00620 respectively (Table S6). Most of the summary statistics including the number of alleles, number of haplotypes, mean size variance and FST or NST values for any population‐pair estimated using the acquired posterior distributions were not statistically different from the observed values (Table S7), suggesting that the scenario‐prior combination of scenario 1 is a good fit. However, mean genic diversity, combined mean genic diversity, number of segregating sites, and mean pairwise differences were underestimated and significantly differed from observed values (Table S7).In DIYABC analysis for the combined mtDNA and microsatellite dataset, scenario 1 was found to be the most likely Fig. , Fig. S4L. amethystinus was supported by high predictive power . The LIG and LGM (CCSM and MIROC) distribution models yielded similar inferences and showed past conditions of suitable habitat somewhat similar to current distribution restricted to mountain ranges . Overall, estimated potential distribution of L. amethystinus indicates that this species had a relatively stable distribution range during the last 140,000 years BP .The current distribution model of  = 0.526, F12, 328 = 7.52, P < 0.0001; exposed culmen, F4, 130 = 4.38, P = 0.002; wing chord, F4, 130 = 13.62, P < 0.0001; tail length, F4, 130 = 12.34, P < 0.0001), in which SMS and TMVB males were larger with shorter bills than those across SMO, TUX, and CHIS . For females, the same pattern of morphological differentiation was observed , with larger females with shorter bills from SMS and TMVB than those across SMO, TUX, and CHIS .For males, the MANOVA of morphological variables followed by univariate ANOVAs showed significant differences among populations grouped by geographic region  between blue‐ and amethyst‐throated groups of populations , and univariate ANOVAs yielded significant group differences for all coordinates in the tetrahedron, hue and chroma variables, except r.vec  when tested against competing scenarios and based on combined mtDNA and microsatellite data. Under this scenario of recent divergence, DIYABC analysis provided good support for an older divergence between groups separated by the IT (CHIS group and the rest of groups) around the end of the LGM , and later divergence between SMS and SMO+TMVB around 3300 years ago, and subsequently between SMO and TMVB groups around 1320 years ago. The DIYABC divergence time estimates supporting postglacial divergence of L. amethystinus groups are very similar to estimates for the joint dataset of mtDNA and microsatellites using IMa, in which convergence was confidently achieved. The reconstruction of genetic histories for recently diverged lineages is hindered by stochasticity in the coalescent process and the confounding influences of migration and incomplete lineage sorting .During the LGM, cloud forest‐adapted species expanded their distribution to the lowlands and their distribution contracted allopatrically during the interglacials according to LGM simulations and the available paleodata for Mesoamerica Graham . The demAmazilia cyanocephala hummingbird species  a star‐shaped haplotype genealogy, (2) demographic expansion and loss of genetic diversity, and (3) marked genetic structuring of populations  a star‐like haplotype network with many low frequency single haplotypes separated from high frequency central haplotypes by few mutational steps, (2) low levels of genetic differentiation between populations, and (3) a mismatch distribution of pairwise differences among haplotypes indicating a sudden increase in expansion from a single population. These signals of expansion from a single population have been reported in the partially codistributed Fs, Tajima's D, mismatch distribution and BSP indicated no significant evidence of population expansions in mtDNA sequences of L. amethystinus. Supporting the dry forest refugia model, the few widespread haplotypes in the networks had frequencies that differed dramatically among the regions. Furthermore, the distribution of haplotype diversity and our geneflow estimates suggest that genetic divergence among groups of populations of L. amethystinus in the corresponding cloud forest areas is due to restricted gene flow across low elevation barriers. This pattern is more complex than that predicted by the dry refugia model, and has been reported in hummingbird‐pollinated Moussonia deppeana  and amethyst (SMO and TMVB) populations after the LGM. The third and final split illustrates a more recent split between SMO and TMVB populations ca. 1300 years ago , deviations from sudden population expansion (SSD and Hri index) or changes in effective population size (BSP) do not satisfy the assumption of panmixia , but the estimated time since divergence between blue‐ and amethyst‐throated populations occurred after the LGM according to IMa and DIYABC analyses. In addition, we found no significant gorget color differentiation between individuals from populations separated for the longest time at the IT according to our divergence time estimates. It is possible that the small amounts of gene flow observed across the IT might erase incipient phenotypic differences, and this gene flow pushed our estimates of divergence times toward present . However, the observed differences between groups of populations in morphometric traits, in which hummingbirds from SMS and TMVB were larger with shorter bills than hummingbirds in localities across the SMO, TUX, and CHIS, do not correspond to the geographically isolated sister groups of populations  or sister groups that differ in throat color . The generally conserved morphometric traits among the sister groups of populations might reflect the ecological similarities between populations separated by lowland barriers.In contrast with male plumage differentiation, we found greater change in morphometric traits of male and female L. amethystinus margaritae) as a separate evolutionary unit, with the assignment of the previously unknown blue‐throated populations from Sierra de Miahuatlán, Oaxaca to L. margaritae instead of L. amethystinus circumventus. Tests of divergence scenarios using multilocus ABC allowed us to reject hypotheses of geographic isolation as a driver for L. amethystinus speciation, but supported a role of refugial‐like dynamics during Pleistocene glacial cycles in generating recent intraspecific genetic structure. We confirmed the importance of geographic barriers such as the Isthmus of Tehuantepec, stressing the need to investigate cryptic barriers and not only conspicuous landscape features such as the IT. Overall, our data support the idea of multiple and varied mechanisms contributing to phenotypic and genetic differentiation of this member of the Mesoamerican biota. The pattern of genetic diversity that we have described herein is likely to be part of a larger phylogeographic signature that was retained as co‐distributed species of the associated cloud forest biota experienced a common history of vicariant events and climatic changes. Understanding the comparative phylogeography of such groups will aid the development and evaluation of phylogeographical hypotheses about the evolutionary history of the Mesoamerican biota and have implications for the conservation of cryptic species.Mitochondrial and nuclear DNA datasets show that the amethyst‐throated hummingbird is composed of two main lineages separated by the IT, congruent with previous finding based on a much smaller sample of mtDNA sequences  from the Dryad Digital Repository : Figure S1. Map of the collection sites of Lampornis amethystinus.Click here for additional data file.Figure S2. Bayesian skyline plots showing means for the historical demographic trends of Lampornis amethystinus groups using mitochondrial sequences.Click here for additional data file.Figure S3. Chronogram based on a Bayesian approach using a coalescent tree prior under and assuming constant population size of Lampornis amethystinus mtDNA sequences in BEAST.Click here for additional data file.Figure S4. (A) Competing demographic scenarios of Lampornis amethystinus divergence and admixture: isolation split model 1  predicts that TMVB (Pop1) merged with SMO (Pop2) at t1 then SMO merged with SMS  at t2 and subsequently with CHIS east of IT (Pop4) at t3; isolation split model 2  is similar to the previous one but predicts that SMS (Pop3) merged with TMVB (Pop1) at t1 then TMVB merged with SMO (Pop2) at t2 and subsequently with CHIS east of IT (Pop4) at t3; isolation with admixture model  consisted of the same basal split between CHIS (Pop4) and the rest of groups west of IT described in previous scenarios but includes a hybridization/lineage fusion event in which SMS (Pop3) is the descendent of admixture between TMVB (Pop1) and SMO (Pop2) at t1, then Pop1 merged with Pop2 at t2, and subsequently with Pop4 at t3.Click here for additional data file.Figure S5. Results from the MAXENT analyses showing species distribution models for Lampornis amethystinus at (a) Last Interglacial , (b) Last Glacial Maximum , (c) Last Glacial Maximum , and (d) at present.Click here for additional data file.Figure S6. Morphological differences between groups of populations of Lampornis amethystinus .Click here for additional data file.Table S1. Collection localities for the Lampornis amethystinus samples examined here.Click here for additional data file.Table S2. Number of genetically analysed samples (n) for the combined ND2 and cytochrome b sequences of Lampornis amethystinus, number of distinct haplotypes (H) found in individuals sampled for each mitochondrial fragment, and the number of individuals per haplotype in parentheses.Click here for additional data file.Table S3. Pairwise comparisons of FST values of mtDNA  and microsatellites  among populations of Lampornis amethystinus grouped based on mountain geography.Click here for additional data file.Table S4. Population genetic variability of groups of populations of Lampornis amethystinus.Click here for additional data file.Table S5. Number of genetically analysed samples for eight microsatellites (n), mean alleles per locus, observed (HO) and expected heterozygosity (HE), and significant deviations of Hardy–Weinberg equilibrium (HWE) for groups of populations of Lampornis amethystinus.Click here for additional data file.Table S6. Model parameters estimated from prior distributions of Scenario 1  using Approximate Bayesian Computation (ABC).Click here for additional data file.Table S7. Comparison of summary statistics for the observed data set and posterior simulated data sets of microsatellites and mitochondrial DNA sequences for Lampornis amethystinus.Click here for additional data file.


Bufo  calamita) populations across the whole of the species’ biogeographical range. Variation at both classes of loci was high in the glacial refugium areas (REF) and much lower in postglacial expansion areas (PGE), especially in range edge populations. Although there was clear evidence that the MHC locus was influenced by positive selection in the past, congruence with the neutral markers suggested that historical demographic events were the main force shaping MHC variation in the PGE area. Both neutral and adaptive genetic variation declined with distance from glacial refugia. Nevertheless, there were also some indications from differential isolation by distance and allele abundance patterns that weak effects of selection have been superimposed on the main drift effect in the PGE zone.Study of major histocompatibility complex (MHC) loci has gained great popularity in recent years, partly due to their function in protecting vertebrates from infections. This is of particular interest in amphibians on account of major threats many species face from emergent diseases such as chytridiomycosis. In this study we compare levels of diversity in an expressed MHC class II locus with neutral genetic diversity at microsatellite loci in natterjack toad ( Xenopus laevis are made up of six exons with an exon-intron organization similar to that of a typical mammalian class II β gene. Exon 2 encodes the β - 1 domain which includes most of the antigen binding sites (ABS) of the beta domain and is the most polymorphic region of the gene The basis of adaptive rather than neutral genetic variation has become increasingly accessible in recent years as loci under selection are identified and characterised. Some of the most popular genes used in this context are those belonging to the major histocompatibility complex (MHC). These genes play an important role in the adaptive immune response of vertebrates. MHC class I molecules present intracellular pathogen peptides to CD8+ T lymphocytes (T cells), primarily in response to viral infections, whereas MHC class II molecules (composed of α and β subunits) present extracellular pathogen peptides to CD4+ T cells after invasion by bacteria and fungi Frequency dependent selection, where bearers of common alleles are likely to be more susceptible to diseases and specific alleles can confer resistance Batrachochytrium dendrobatidis  with reverse primer 2R307b (ATAATTCAGTATATACAGGGTCTCACC) amplified a sequence of 279–282 base pairs (excluding primers). The 20 µl PCRs contained approximately 25 ng DNA, 0.4 µM of each primer, 100 µM dNTPs, 1x reaction buffer and 0.5 U of New England Biolabs Taq DNA polymerase. Thermal cycling consisted of an initial denaturation step of 94°C for 3 min and a touchdown protocol with a total of 35 cycles and ending with a elongation step of 72°C for 10 min. Each cycle started with a denaturation step of 94°C for 30 sec and ended with an elongation step of 72°C for 40 sec. Annealing temperatures consisted of 2 cycles at 62°C, 2 cycles at 60°C, 2 cycles at 58°C and 29 cycles at 56°C, each for 30 sec. Individual alleles were identified by SSCP analysis as described in Zeisset & Beebee et al. MHC class II locus B β chain exon 2 sequences of ST outlier approach implemented in LOSITAN For comparative purposes we used microsatellite data obtained in previous studies from eight polymorphic loci in 600 individuals sampled from 18 populations B. calamita locus B allele ‘Buca B2’ and used NNSPLICE version 0.9 http://www.fruitfly.org/seq_tools/splice.html, to predict splice sites.To determine intron/exon boundaries we combined the putative intron and exon sequences (Genbank nos.: JX258913 and JX258914) to obtain a 532 base pair sequence of et al. Sequences were aligned and edited manually using Bioedit v. 7.0.9 http://www.datamonkey.orgB.calamita, Bufo bufo and Bufo  viridis . After testing for recombination, a phylogenetic tree was inferred and used as the input for selection on particular codons using the two methods.To identify specific sites under selection we used two new methods: a mixed effects model of evolution (MEME) to identify instances of both episodic and transient positive selection at individual sites Bufo species we used three methods were to test for signatures of recombination. This analysis was carried out for 282 bp of exon 2 sequence as well as for the 157 bp we used in phylogenetic tree reconstruction to investigate the effects recombination may have on tree construction. For this analysis we also included B. calamita, B. bufo and B. viridis locus A alleles  in each population was assessed for microsatellite and MHC loci by applying the exact tests in Genepop 4.0.10 E) and allelic richness , were estimated for each population and overall in FSTAT 2.9.3.2 ST primarily as a way to measure the level of differentiation between populations. As FST may be affected by highly variable markers such as microsatellites we also calculated DESTST and DEST within and between each class of loci, as well as isolation by distance (using ln distance vs. FST/1−FST), were made using the Mantel test facility in Genepop with 1000 permutations. Distances were measured using Google Earth between all sampling sites north of the Pyrenees  that were not separated by sea, as most amphibians cannot survive seawater exposure. Distances were otherwise direct (Euclidean) allowing for bends to avoid sea water where necessary.F-statistics To investigate how variation was partitioned within and among REF and PGE populations we carried out an AMOVA in Arlequin v. 3.5 Phylogeographic relationships among the populations based on allele frequencies were determined separately for microsatellite and MHC data using Phylip v. 3.66 E) between REF and PGE populations as well as correlations between microsatellite and MHC data and microsatellite and allelic richness (R) and geographic distance were carried out using Statistix7 .Standard statistical tests for differences in allelic richness (R) and expected heterozygosity . Using MEME (P<0.1) we identified 23 positively selected codons in exon 2 and using FUBAR with a posterior probability >90% we found 19. Codons identified by both methods largely corresponded to putative antigen binding sites (ABS) as defined by Brown et al. et al. B. calamita. Average amino acid distance at this locus was 0.149 (SE 0.028). Nucleotide and amino acid distances were much higher in the putative ABS  than in non-ABS  sites.Splice site analysis for locus B predicted intron/exon boundaries between base pairs 3 and 4 after the 3′ end of the forward primer binding site and between base pairs 281 and 282 (2 bp into the reverse primer binding site), making the putative exon 279 base pairs long. These sites corresponded to exon 2 boundaries found in some other amphibian species B. calamita, ten showed evidence of a recombination event including a codon insertion towards the 3′ end resulting in the addition of threonine. Five had a deletion of three nucleotides which corresponded to a codon deletion in the MHC class II DAB alleles of the great crested newt, Triturus cristatus  from locus B in 17 populations of Recombination tests GeneConv and MaxChi detected between two and three recombination events among all the whole exon 2 MHC sequences, with between five and 15 recombination signals. GARD on the other hand detected no evidence of recombination. For the 157 bp sequences used in phylogenetic tree reconstruction only MaxChi detected one recombination event, with 13 recombination signals.Rana, Xenopus and Discoglossoidea (Bombina and Alytes species) sequences all formed separate groups. Within those groups there was also strong support for some branches separating species. The Central American Sachatamia and Espadarana clustered strongly with the European Bufo. Both the phylogenetic network  in the MHC data, each of which was found in a single population . Twenty Twenty-four of 126 population × microsatellite loci comparisons showed significant deviations from HWE after Bonferroni correction. In all cases this was due to a homozygote excess and Micro-Checker indicated the possibility of null alleles in several populations at Bcalµ1, 2, 3, 4 and 6 with estimated frequencies ranging from 0.09 to 0.32 . The numAfter Bonferroni corrections there were no cases of linkage disequilibrium among the loci.2 = 4.20, DF = 1, P = 0.0404; microsatellites: χ2 = 6.92, DF = 1, P = 0.0085). MHC and microsatellite allelic richness was significantly correlated among populations where both types of loci were sampled . Microsatellite and MHC allelic richness outside Iberia declined with distance from the SW France refugium area . Expected heterozygosity between the types of loci was also significantly correlated . None of the correlations were unduly influenced by populations with a significant amount of null alleles . Average FIS values of MHC and microsatellite loci across all populations in this PGE zone were low in both cases (0.019 and 0.028 respectively) and not significantly different .For both microsatellite and MHC markers most variation was accounted for at the within-population level (49% and 42% respectively) or among populations within REF and PGE groups . However, in several cases where geographical separation was high, MHC DEST = 1 thus providing incomplete resolution of differentiation level. Subsequent comparisons therefore focused on pairwise FST estimates which were correlated between MHC and microsatellite loci . Excluding Iberian populations, among areas analysed for both microsatellites and MHC genotypes and not separated by seawater , the correlation between MHC and microsatellite pairwise FST estimates was also strong . Mean pairwise FST estimates in this region were similar for both types of loci  and there was significant isolation by distance . However, the strength of IBD was greater for MHC than for microsatellites as shown using untransformed FST and distance estimates in ST estimates are expected to be greater than those on MHC loci, due to their higher mutation rates.Pairwise Fdata see . Using MPhylogeographic trees based on microsatellite and MHC allele frequencies were broadly congruent . HoweverRana warszewitschii to 0.155 in R. catesbeianaet al. et al. Bufo MHC class II loci. Similar findings have been reported by others  were comparable to those found in other amphibian species, where diversity ranged from 0.062 in ers e.g. , indicatB. calamita MHC were alleles  that had a codon deletion at the same position as found in the great crested newt Triturus cristatus in Romanian REF populations Espadarna prosoblepon  in post glacial expansion (PGE) areas possessed a subset of alleles from the refugia (REF) populations, when they compared three PGE populations from across Europe to only four small REF populations from Romania. Our data support the theory that natterjack toads survived the Weichselian glacial maximum 20 000 years BP in at least one north European refuge, most likely in France, and colonized northern Europe from there B. calamita and T. cristatus variation of microsatellite and MHC loci was high in the REF groups and much lower in the PGE groups. A decrease in allelic diversity from southern to northern Europe is well documented in neutral loci , not much different from that of the microsatellites. Microsatellite mutation rates increase with microsatellite length and contractions become more likely than expansions as length increases When comparing two different marker systems such as MHC loci and microsatellites it is important to consider the potential differences in the ages of observed alleles due to the higher mutation rates of microsatellites and potential back mutations. Microsatellite mutation rates in B. calamita MHC variation across the species’ range there were some interesting differences between MHC and microsatellite genotype distributions that may imply an additional, albeit minor role of selection in structuring the MHC locus at this large geographical scale. MHC alleles were more sharply differentiated between the REF and PGE regions than were the microsatellite alleles. Isolation by distance effects in those parts of the mainland Europe PGE area where gene flow remained possible after postglacial sea level rise was significantly stronger for the MHC than for the microsatellite loci. The commonest MHC alleles in the PGE group were, on average, at higher frequency and more geographically clustered than the commonest alleles in the microsatellite locus with the most comparable allelic diversity. This pattern difference might imply weak effects of selection reflected in patterns of common MHC allele abundance in specific regions, such as north-west France, north-central Europe and eastern Europe, perhaps in turn reflecting local differences in pathogen communities. Pathogens often exhibit a ‘latitudinal diversity gradient’, with high diversity at the equator decreasing towards the poles B. calamita is maintained by higher pathogen- mediated selection pressure, but further studies into actual difference of pathogen prevalence in the various regions are needed to test this hypothesis.Despite the likely dominance of drift effects on B. calamita MHC locus at the biogeographical scale. This is essentially similar to the situation discovered with a comparable study of another widespread European amphibian, Triturus cristatusClupea harengus) also failed to detect any evidence of selection acting on the MHC locus, although in this study a MHC-embedded microsatellite locus was used and it is not clear to what extent this reflected variability in the exon et al.B. bufo population translocated over 400 km within the UK adapted within three generations to an allele frequency distribution similar to that of neighbouring populations in the receptor area Myodes glareolus) found no spatial genetic structure among populations at MHC loci, but clear differentiation reflecting their major glacial refugia at a mtDNA gene. This may indicate yet another situation in which spatiotemporal variations in selection pressures acting over large areas can mask historical signals of population origins Overall, our evidence clearly implies a stronger influence of drift than selection on this Figure S1Phylogenetic network of MHC class II beta exon 2 sequences. Neighbor-Net tree based on Jukes-Cantor distances of 282 bp of sequence of MHC locus B from B. calamita.(TIF)Click here for additional data file.Table S1ST values.Population differentiation as estimated by F Numbers in brackets refer to population numbers in ST values for MHC data. P-values obtained after: 2720 permutations. Indicative adjusted nominal level (5%) for multiple comparisons is: 0.000368. B: Population differentiation as estimated by FST values for microsatellite data, based on 7 loci. P-values obtained after: 3060 permutations. Indicative adjusted nominal level (5%) for multiple comparisons is: 0.000327.(XLSX)Click here for additional data file.File S1Table A (sheet 1): Allelic frequencies in each population for microsatellite and MHC loci. Numbers in brackets refer to population numbers in (XLSX)Click here for additional data file.