High rates of unintended pregnancies worldwide indicate a need for more accessible and acceptable methods of contraception. We have developed a monoclonal antibody, the Human Contraception Antibody (HCA), for use by women in vaginal films and rings for contraception. The divalent F(ab’)2 region of HCA binds to an abundant male reproductive tract-specific antigen, CD52g, and potently agglutinates sperm. Certain other antibody activities mediated by the Fc region such as mucus trapping, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) could have beneficial or negative effects. The purpose of this study was to document HCA Fc effector functions and determine whether an engineered variant of HCA with a modified Fc region, HCA-LALAPG, retains desirable contraceptive activity while minimizing Fc-mediated effects. Fab and Fc functions were compared between HCA and HCA-LALAPG. Fab activity was assessed using sperm agglutination and modified swim-up ("sperm escape”) assays. Fc functions were assessed by CDC (sperm immobilization), ADCP, and cervical mucus penetration assays. HCA and HCA-LALAPG showed equivalent activity in assays of Fab function. In the assays of Fc function, HCA supported strong CDC, ADCP, and sperm trapping in cervical mucus whereas HCA-LALAPG demonstrated little to no activity. HCA and the HCA-LALAPG variant were both highly effective in the sperm agglutination assays but differed in Fc mediated functions. Use of the HCA-LALAPG variant for contraception in women could reduce antibody-mediated inflammation and antigen presentation but may have reduced contraceptive efficacy due to much weaker sperm trapping in mucus and complement-dependent sperm immobilization activity. The United Nations reports that almost half of all pregnancies are unintended due to lack of or incorrect use of effective contraception . This poNicotiana-based production platform to produce monoclonal antibodies (mAbs) for reproductive health. Our group recently tested a multipurpose prevention technology (MPT) product, a vaginal film containing Nicotiana-based mAbs against human immunodeficiency virus 1 (HIV-1) and herpes simplex virus type 2 (HSV-2), in a Phase 1 clinical trial. The film, MB66, was safe and maintained antiviral activity in the vaginal mucosa for up to 24 hours [Over 100 antibody-based drugs have been approved by the US Food and Drug Administration (FDA) with many more currently in clinical trials . While aThe majority of mAbs produced for clinical applications, including our lead HIV-1, HSV-2, and HCA mAbs, belong to the IgG1 antibody subclass . IgG1 mAFc-mediated antibody functions are desirable for certain applications such as mAb-mediated pathogen clearance, but they may be undesirable in other situations. ADCC and CDC can elicit inflammation , and ADCAs part of clinical development, various engineered versions of HCA are being tested to determine whether they enhance antibody potency and reduce potential side effects. HCA variants have been engineered with increased valency to enhance agglutination potency . A LALAPNicotiana benthamiana from the variable region sequence of H6-3C4 [Nicotiana plants with knockdown of fucosyl- and xylosyl-transferases allowed for the insertion of humanized glycans as previously described [Nicotiana mAb platform from a DNA template with three amino acid substitutions in the Fc region: L234A, L235A, and P329G.HCA, a human anti-CD52g mAb, was produced in f H6-3C4  and an oescribed . HCA-LALCampath-1, or alemtuzumab, is a humanized IgG monoclonal antibody used for cancer immunotherapy . Campath-1 binds to a peptide epitope present on CD52, an antigen found on normal and malignant hematopoietic cells, and CD52g, a heavily glycosylated male reproductive tract-specific form of CD52. HCA binds to a carbohydrate epitope only found on CD52g. Campath-1 was used as a positive control for the sperm phagocytosis assay as it has been shown to bind to sperm .Nicotiana. It was used as an isotype control in the sperm phagocytosis assay.VRC01-N, a broadly neutralizing anti-HIV-1 IgG1 antibody , was alsThis study was approved by the Institutional Review Board at Boston University Medical Campus (Human Subjects Protocols H36843 and H41454). All participants provided written informed consent prior to participation.6/mL for use in the assays.Human semen samples were obtained from healthy men aged 18–45 after at least two days of sexual abstinence. All samples met the WHO criteria for fertility . SamplesMidcycle cervical mucus was collected by a trained physician from reproductive-aged women within 48 hours of a positive ovulation test . An endocervical pipelle  was used to aspirate pure cervical mucus directly from the endocervical canal. None of the women were on hormonal contraception. Ovulation was confirmed for all women by evaluation of the mucus (clear with spinnbarkeit characteristics), and by estrogen and progesterone levels in blood. Cervical mucus was diluted 1:3 in PBS and aspirated into flat capillary tubes . Both ends of the tube were sealed with parafilm, and samples were stored at 4°C for up to 5 days. Cervical mucus samples from four different donors were used for this study.The effects of HCA and HCA-LALAPG on sperm agglutination were determined using the sperm agglutination kinetic assay as described previously . A 2 μL The percentage of progressively-motile sperm that escaped sperm agglutinates after antibody exposure was quantified as described previously . Sperm wThe sperm immobilization test was performed as described previously , 31, 32.6/mL in seminal plasma instead of MHM. Antibodies were diluted to 12.5 μg/mL in MHM. Ten μL of diluted antibody or MHM alone (control) were added to one end of the capillary tube, and the opposite end was then sealed with parafilm. The open end of the capillary tube was inserted horizontally into a tube containing 100 μL of sperm so that the sperm would swim through the antibody or medium (control) before entering the column of cervical mucus. Tubes were incubated in a humidified chamber at 32°C. At 30-, 60- and 90-minute time points, sperm were assessed at 1, 2, 3 and 4 cm depths using the CASA. Progressive sperm in each section were scored by manual review of the CASA output by a trained researcher.Capillary tubes containing cervical mucus were warmed to 32°C and each 1 cm interval on the capillary tube was marked. Motile sperm were prepared as described above but re-suspended at a concentration of 25–40 x106 cells/mL in 3 mL of RPMI 1640 complete medium . Cells were treated with 100 ng/mL phorbol-12-myristate-13-acetate (PMA) to induce differentiation into macrophages [2 at 37°C for 48–72 hours, after which they became adherent to the coverslips.U937  pro-monocytes were seeded onto sterile glass coverslips in 6-well plates at a density of 0.5 x 10rophages  and were6 sperm in either MHM or in MHM supplemented with 50 μg/mL antibody were added to each well. Following a 30-minute incubation at 37°C and 5% CO2, the macrophage cultures were washed 3X with PBS. After another 30-minute incubation at 37°C in PBS, the macrophage cultures were incubated in trypsin for 5 minutes to remove non-specifically bound spermatozoa. The coverslips were treated with Differential Quik III fixative solution , stained with Differential Quik III stain solution I and II, and washed in tap water before being mounted to glass slides. Macrophages were observed under a light microscope at X200 magnification, and the number of spermatozoa associated with 100–300 macrophages was counted for each treatment group. Number of internalized sperm per macrophage was also assessed and characterized as sperm partially engulfed, either head or tail, or wholly engulfed by a macrophage. The experiment was repeated three times using semen samples from different donors.The sperm phagocytosis assay was performed as described by Oren-Benaroya et al. with modifications . In brieData were analyzed by either repeated measures analysis of variance (ANOVA) or mixed-effects analysis. A significant analysis was followed by either post hoc Tukey or Sidak multiple comparison tests as indicated by the type of analysis. If continuous variables were not normally distributed, they were log  transformed prior to analysis. The equality of variances of the pairwise differences of within subject conditions was assessed through a test of sphericity (Geisser-Greenhouse’s epsilon). GraphPad Prism  and JMP Pro  were used for statistical analysis and graph creation. Differences were considered to be statistically significant when p<0.05.Because agglutination of sperm relies on the Fab portion of HCA, Fc mutations would not be expected to impact agglutination potency. We compared the agglutination function of parent HCA and HCA-LALAPG using agglutination kinetics and sperm escape assays. As expected, the two antisperm antibodies did not differ in time to 100% agglutination over a wide concentration range p > 0.6, . In factHCA significantly immobilized sperm in the presence of complement; sperm motility was significantly reduced at HCA concentrations ranging from 12.5 to 3.12 μg/mL  since antigen presentation of sperm antigens could lead to antisperm immunity and infertility. Differentiated U937 macrophage-like cells were incubated for 30 minutes with antibody-treated sperm, after which they were washed for 30 minutes in PBS and treated with trypsin for 5 minutes to remove non-specifically bound spermatozoa. Fixed slides were stained with Diff Quik stain kit, a morphology dye stain, and observed under a light microscope to quantify the number of associated spermatozoa per macrophage-like cell. Associated spermatozoa were characterized as those that were either attached to the surface of a cell , or partially engulfed. The positive control antibody, Campath, elicited the greatest level of sperm association with more than one spermatozoon per effector cell . The numA phagocytosis time-course experiment was conducted to determine whether there was an effect of incubation time on the number of HCA-coated sperm that were phagocytosed by U937 macrophages. Internalized sperm were scored as either partially (head or tail) or wholly phagocytosed. The number of internalized spermatozoa per macrophage increased over time  and reacHCA is an antisperm IgG monoclonal antibody currently being studied in preclinical and clinical studies for its contraceptive activity. Here, we characterized an Fc-engineered variant (HCA-LALAPG) to delineate Fab and Fc-mediated functions and to evaluate an alternate vaginal film candidate that may reduce undesirable inflammatory and immune side effects. No differences in sperm agglutination potency were detected. Agglutination, mediated by the Fab region, is thought to be the main contraceptive mechanism of HCA. Both HCA and HCA-LALAPG rapidly agglutinated sperm within 60 seconds  even at Differences in Fc-mediated functions between HCA and HCA-LALAPG were observed. Some antisperm antibodies can immobilize sperm in the presence of a complement source , 31. We The ability of HCA and the HCA-LALAPG variant to trap sperm in cervical mucus was also compared as this mechanism is thought to contribute to the HCA contraceptive effect. While the exact mechanism of mucus trapping is not fully understood, antibodies may bind to cervical mucus through sugar-sugar hydrogen bonds or Fc receptor-like molecules , 39; othLastly, we demonstrated that HCA, but not HCA-LALAPG, mediates sperm phagocytosis by macrophage-like cells. The ability of HCA to mediate ADCP was previously unknown. The inability of HCA-LALAPG to elicit ADCP was unsurprising since this effector function is dependent upon FcγR binding. HCA-mediated sperm phagocytosis may have serious implications in the context of the female reproductive tract (FRT). Macrophages are widely distributed in the FRT and fluctuate with changes in hormone levels throughout the menstrual cycle . These pAntibody engineering technology continues to improve, offering greater possibilities for antibody design and customization. The challenge is determining the optimal antibody profile in the desired context, especially one as specialized as the vaginal mucosa. Understanding the potential Fc effector functions of an antibody-based drug is critical to the development of a safe and effective product. We have established that the HCA-LALAPG variant had decreased Fc-mediated functions compared to HCA. Some Fc functions such as mucus trapping and sperm immobilization may contribute to the contraceptive efficacy of HCA and may be desirable. Other Fc functions such as ADCP may promote detrimental immune responses through antigen presentation and host production of antisperm antibodies. Fc-engineered variants of HCA may provide safer, more conservative alternatives to HCA, but results from this study indicate that HCA-LALAPG may not be optimal for this role. Use of high resolution maps of FcR binding sites on human IgG1  could prS1 FigThe number of progressively motile sperm at 30 minutes (A), 60 minutes (B), and 90 minutes (C) in ovulatory cervical mucus, at capillary tube depths of 1, 2, 3 and 4 cm. The number of progressive sperm was significantly lower in the HCA treatment group than with HCA-LALAPG at all time points. Statistics are given in (TIF)Click here for additional data file.S2 FigThree out of four donors showed far fewer progressive sperm in the HCA treatment group compared with HCA-LALAPG and medium-only treatment  while Donor 104 had fewer progressive sperm in any of the treatment groups .(TIF)Click here for additional data file.S3 FigThe number of internalized sperm per macrophage increased in HCA-treated cultures overtime; a significant increase was observed between 1 hour and 2.5-hour time points (p = 0.018). HCA-LALAPG, medium-only control, and isotype control (VRC01) did not mediate sperm phagocytosis even at the 2.5-hour time point. Final antibody concentrations were 50 μg/mL. Data are expressed as mean ± SEM of three independently performed experiments. Statistical analyses were conducted using repeated measures one-way ANOVA of log-transformed data followed by Tukey multiple comparisons tests.(TIF)Click here for additional data file.S1 TableA) Number of progressively motile sperm in the cervical mucus penetration test (shown as mean ± SEM). B) Statistical differences between treatment groups at each distance. Significant comparisons indicated by shaded cells.(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Orchidaceae Juss. of the northern part of Kazakhstan, within the steppe, forest-steppe and semi-desert habitats of the country . The investigation is based on herbarium materials, literature data and field observations. We examined material from the following herbarium collections: LE, MW, TK, MHA, SVER, KUZ, ALTB, AA, NUR, KG, KSPI, NS, NSK, MOSP, ORIS, PPIU, totalling 288 herbarium specimens. The paper presents data in the form of revision, focusing on orchids of the northern part of Kazakhstan. It is accompanied by maps indicating localities, notes on habitat preferences, phenology and conservation status. A total of 25 species of 16 genera were recorded, of which eight are included in the Corallorhizatrifida, Epipactisatrorubens, Gymnadeniaconopsea, Hammarbyapaludosa, Herminiummonorchis, Liparisloeselii, Malaxismonophyllos, Neottiacamtschatea and Spiranthesaustralis. The most widespread species in the studied region are Dactylorhizaincarnata, D.umbrosa and Epipactispalustris. The rarest species  are Epipactisatrorubens, E.helleborine, Epipogiumaphyllum, Hammarbyapaludosa and Herminiummonorchis.We present a review of  Orchids are one of the largest families in the world, numbering, according to various data, from 28,000 to 30,500 species . Due to Orchidaceae Juss. in Kazakhstan is low due to the prevalence of an arid climate with a rather harsh temperature regime in the cold period. According to the last vascular plant list of Kazakhstan by Cypripedium×ventricosum Sw. (Epipactisatrorubens (Hoffm.) Besser  Kuntze, Neottiacordata (L.) Rich.  Besser , Hammarb.) Rich. .In recent years, the study of orchids of Kazakh Altai, which accounts for 22 species, has received particular attention : Aktyubinsk, Bukeev, Emba, Eastern Upland, Irtysh, Karkaraly, Kokchetav, Mugodzhary, Priaral, Prikaspiy, Semipalatinsk Pinery, Syrt, Tobol-Ishim, Turgay, Ulutau and Western Upland  and the herbarium of Zhezkazgan Botanic Garden, Zhezkazgan, (termed “ZhBG”). All herbarium collections were studied either personally or after photographs.We have studied the following herbarium collections: LE, MW, TK, MHA, SVER, KUZ, ALTB, AA, NUR, KG, KSPI, NS, NSK, MOSP, ORIS and PPIU  are included on the maps as well.According to our data, 25 species of orchids from 16 genera are recorded in the northern part of Kazakhstan. Eight species are listed in the Red Data Book of Kazakhstan, of which four species are classified under the II category and four species under the III category.The distribution of the studied species within the floristic regions is as follows: Kokchetav – 14 species, Tobol-Ishim – 13 species, Irtysh – 9 species, Mugodzhary – 8 species, Eastern Upland – 8 species, Karkaraly – 8 species, Semipalatinsk Pinery – 7 species, Aktyubinsk – 2 species, Syrt – 3 species, Western Upland – 3 species, Ulutau – 1 species, Emba – 1 species and Prikaspiy – 1 species , is located in the semi-desert zone of Kazakhstan; however, the Urkach and Ber-Chugur places  are located here, which include extensive lowlands with birch-aspen forests and sphagnum swamps, a very rare type of habitat in Kazakhstan. The Urkach place is considered to be a unique remnant of fragments of northern vegetation that retreated to the north during dry interglacial times and are evidence of the former vegetation of the Mugodzhar Mountains  was found, as well as Turgay FR and Priaral FR, where orchids were not found at all, represent desert and semi-desert zones of Kazakhstan, with high soil salinity. The small number of orchids in Ulutau FR  Soó , inThe distribution of the studied species by administrative regions is the following: Akmola – 12 species, Pavlodar – 13 species, Karaganda – 10 species, Kostanay – 10 species, East Kazakhstan – 8 species, North Kazakhstan – 7 species, West Kazakhstan – 3 species and Aktobe – 8 species  Soó  Crantz  Rich  R.Br.  R.Br., Liparisloeselii (L.) Rich., Malaxismonophyllos (L.) Sw., Neottiacamtschatea (L.) Rchb. f. and Spiranthesaustralis (R.Br.) Lindl. Thus, 17 species of orchids growing in the studied region should be included in the next edition of the Red Book of Kazakhstan.Currently, eight species of orchids growing in the northern part of Kazakhstan are included in the  L. Figs , 4B, C.gSw. Figs , 4D, DacSoó Figs , 5A, Epintz Figs , 4G, EpiSw. Figs , 4H, Orcich Figs , 6I. We el. Figs , 4A, EpiBr. Figs , 6A, HamFR. In our assessment, it appears to be Dactylorhizaochroleuca (Wüstnei ex Boll) Holub. However, this finding requires confirmation, as there is a possibility of confusion with hypochromic variants of Dactylorhizaincarnata. Dactylorhizasibirica Efimov , Epipactispalustris , Dactylorhizaumbrosa (Kar. & Kir.) Nevski . The rarest species (one location only) are Epipactisatrorubens, Epipactishelleborine (L.) Crantz, Epipogiumaphyllum, Hammarbyapaludosa and Herminiummonorchis  P.F. Hunt & Summerh and D.russowii (Klinge) Holub, reported earlier for the studied region (Dactylorhiza×kerneri (Soó) Soó (= D.fuchsii × D.incarnata) for the flora of Kazakhstan, based on herbarium collections stored in LE. We believe that those specimens can be rather safely determined as Dactylorhizasibirica, a hybridogenous species originating according to the same hybrid formula.d region , are excTaxon classificationPlantaeAsparagalesOrchidaceaeChâtel.50691C44-7BF3-5F06-9F99-210F72106BD2Russia , Kazakhstan .Akmola Region: Sandyktau District: Maraldy, near the village of Sandyktau, 5 Jul 1913, Semenov s.n.. (TK!); Burabay District: “Burabay” State National Nature Park: east shore of Shchuchye Lake, 17 Jun 1972, Gorchakovskiy s.n.. (SVER 695750!); upper reaches of Imanaevskiy Spring, 2 Aug 1895, Gordiagin 594 (LE!); near Burabay, 12 Jun 1913, Drobov 430 (LE!); near Karas’e Lake, 27 Jun 1901, Gordiagin 514 (LE!); same loc., 20 Jun 2012, Khrustaleva and Artemova s.n.. (KUZ 02684!); near of Svetloe Lake, 15 Jul 1960, Denisova 1577 (MW 0816955!); Aiyrtau District: Kokshetau State National Nature Park, Imantau Mountains, “Buyan-Schel” place, 31 May 1973, Gorchakovskiy s.n.. (SVER 695749!). Karkaraly: Karaganda Region: Karkaraly District: Karkaraly Mountains, Alexandrov Klyuch cordon, 31 May 2007, Kupriyanov et al. s.n.. (KUZ 11464!).Kokchetav: Forest swamps, stream valleys, lakesides, sphagnum swamps and wet birch forests.Flowering in May–Jun; fruiting in Jul–Aug.Not protected. We recommend to include this species in the next edition of the Red Book of Kazakhstan.Corallorhizatrifida is reported for the Kokshetau State National Nature Park for the first time.Taxon classificationPlantaeAsparagalesOrchidaceaeL.3C107815-3A16-5E80-8BE8-93BAC661D5CCRussia , Kazakhstan (Altai).Akmola Region: Burabay District: “Burabay” State National Nature Park: near Shchuchinsk, Barmashinskoe forestry, 12 Jul 2019, Kubentaev s.n.. (NUR!); same loc., 15 Jun 1965, Oleneeva and Antoshenko s.n.. (SVER 695751!); same loc., 18 Jun 2012, Artemova s.n.. (KUZ 02637!); same loc., 8 Jun 2011, Kupriyanov and Hrustaleva s.n.. (KUZ 01096!); same loc., 2.5 km northeast of Shchuchinsk, 12 Jun 2011, Hrustaleva s.n.. (KUZ 00884!); Zolotoborskoe forestry, 23 Jun 2016, Hrustaleva and Artemova s.n.. (KUZ 02798!); same loc., 3 km south of the Barmashino, 13 Jun 1972, Gorchakovskiy s.n.. (SVER 695752!); near Barmashino Lake, 26 Jun 1890, Gordyagin 503 (LE!); the northern shore of Kotyrkol Lake, 19 Jun 1902, Ignatov and Petrovskiy 209 (LE!); same loc., 31 May 1902, Ignatov and Petrovsky 209 (LE!); located 3–3.5 km east of Katarkol ; on the right shore of Ishim River, near Krasnoyarka, Jun 1968, Syzganov et al. s.n.. (NKU!); same loc., Jun 1968, Terekhina et al. s.n.. (NKU!); same loc., Jun 1968, Freze s.n.. (NKU!). Irtysh: Pavlodar Region: Zhelezinskiy District: on the right shore of Irtysh River . It is a rare and endangered species protected in the “Burabay” State National Nature Park, “Sogrov” State Nature Reserve and “Floodplain of the Irtysh River” State Nature Reserve.Cypripediumcalceolus are located near Shchuchinsk and the village of Burabay, in areas with high recreational activity. These populations require special attention and protection due to the low number of plants in the populations, which can be attributed to the significant anthropogenic impact in these areas , Kazakhstan (Altai).North Kazakhstan Region: Kyzylzhar District: on the right shore of Ishim River, approximately 75 km north of Petropavlovsk and 5 km north of Krasnoyarka, 16 Jun 1968, Syzganov and Sadvokasova s.n.. (LE); the left shore of Ishim River, near Krasnoyarka, 17 Jun 1968, Sidarkina and Galieva s.n. (NKU!); near Vagulino, 12 Jun 1982, Rain and Martyasheva s.n.. (NKU!); near Tashkentka, 25 Jun 1982, Vafina et al. s.n. (NKU!). ? Kostanay Region: ?Uzynkol District .Tobol-Ishim: District , ?MendykDistrict . Irtysh:Wet birch forests.Flowering in Jun; fruiting in Jul–Aug.This rare species is included in the Red Book of Kazakhstan (category II) and is protected within the territory of two State Nature Reserves: “Sogrov” and “Floodplain of the Irtysh River”.Cypripediumguttatum for the Kostanay Region is doubtful since we have not found herbarium collections from these areas, including the herbarium of Kostanay Pedagogical University (KSPI), where the Pugachev collections are stored.The report of Taxon classificationPlantaeAsparagalesOrchidaceaeSw.40291F74-E7A4-5866-9CA2-C5A7CE538186Russia , Kazakhstan (Altai).North Kazakhstan Region: Kyzylzhar District: on the right shore of Ishim River, 5 km north of Krasnoyarka, 16 Jun 1968, Kolodchenko s.n.. ; on right shore of Ishim River, near Krasnoyarka, Jun 1968, Shakarova et al. s.n.. (NKU!); same loc., 27 Jun 1987, Samoylova et al. s.n.. (NKU!). ?Kokchetav: ?Akmola Region [without detailed locality] . Semipalatinsk Pinery: East Kazakhstan Region: Beskaragay District: near Kara-Murza, 16 Jun 1956, Olovitikova s.n.. (LE!).Tobol-Ishim: ocality] . Irtysh:Birch forests and valleys of forest streams.Flowering in Jun; fruiting in Jul–Aug.This very rare species is included in the Red Book of Kazakhstan (category II). It is protected within the “Sogrov” and “Floodplain of the Irtysh River” State Nature Reserves.Cypripediummacranthos within the territory of Kokchetav FR.According to recent reports  and our C.×ventricosum  hybrid was found: “Tobol-Ishim: North Kazakhstan Region: Kyzylzhar District: on the right shore of the Ishim River, near Krasnoyarka, 17 Jun 1968, Tsykareva s.n.. (AA!)”. This species occurs in areas where parent species co-occur, forming transitional populations with intermediate morphology  SoóA94DE8F2-832B-5B2D-A277-E3823F7C3CF6Dactylorhizahebridensis (Wilmott) Aver., ≡Dactylorhizafuchsiisubsp.hebridensis (Wilmott) Soó).  , Kazakhstan (Altai).Akmola Region: Burabay District: Near Karas’e Lake, 4 Jul 1937, Shishkina s.n. (AA!); same loc., Zolotoborskiy forestry, 21 Jun 1972, Gorchakovskiy s.n.. (SVER 695767!); Borovskoy forest area, upper reaches of Imanayevsky Creek, 27 Jun 1974, Gorchakovskiy s.n.. (SVER 695769!); same loc., 16 Jun 1972 Gorchakovskiy s.n.. (SVER 695769!); Barmashinskiy experimental forestry, 1 Aug 1965, Tyulebergeneva s.n. (SVER 695763!); Zolotoborsky forestry, eastern shore of Shchuchye Lake, 3 km north of the Medvezhiy cordon, 17 Jun 1972, Gorchakovskiy s.n.. (SVER 695756!); Burabay forest area, near Akylbay cordon, 10 Jun 1913, Drobova 308 (LE!); Burabay forest area, near Dorofeyevka, 10 Jun 1913, Drobova 309 (LE!); “Burabay” State National Nature Park: Borovskoe forestry, 16 Jun 2019, Kubentayev and Alibekov s.n.. (NUR!); Barmashinskoe forestry, 7 Aug 2020, Kubentayev et al. s.n.. (NUR!); Temnoborskoe forestry, near Zhukey Lake, north-west coast, 11 Jun 2019, Kubentayev et al. s.n.. (NUR!); near Burabay, the headwaters of Imanayevsky Creek, 7 Jun 2011, Kupriyanov s.n.. (KUZ 00816!); near Makinka, 11 Jun 2011, Kupriyanov s.n.. (KUZ 01111!); Zerendi District: “Kokshetau” State National Nature Park: Zerendi forestry, near Zerenda, 28 May 2020, Kubentayev and Alibekov s.n.. (NUR!); Oramndybulakskoe forestry, near Karsak, 27 Jun 2020, Kubentayev and Alibekov s.n.. (NUR!); Zerendi forestry, near Krasniy cordon, 26 Jun 2019, Kubentayev et al. s.n.. (NUR!); Sandyktauskiy District: Sandyktau forestry, near “Chernichniy log”, 16 Jun 1957, Gribanov s.n.. (AA!). North Kazakhstan Region: Aiyrtau District: Imantau Mount, 7 Aug 1965, Oleneva and Antoshenko s.n.. (695765 SEVR!); same loc., the valley of the stream flowing down from Imantau Mount, 30 May 1973, Gorchakovskiy s.n.. (SEVR 695760!); Imantau Mount, Bayan Gorge, 28 Aug 1981, Gorchakovskiy s.n.. (SEVR 627680!); “Kokshetau” State National Nature Park, near Syrymbet, 10 Aug 2020, Kubentayev et al. s.n.. (NUR!); near Lobanovo, 27 Jul 2019, Kubentayev et al. s.n.. (NUR!). Tobol-Ishim: Kostanay Region: Mendykara District: botanical nature monument “Planting of birch and pine forests near Borovskoye Lake”, Jun 2009, Perezhogin ; Denisovskiy District: Ordzhonikidzevskoye forestry, near Denisovka (KSPI students (SVER 507474!). Karkaraly: Karaganda Region: Karkaraly Mountains, 27 Jun 1843, Schrenk s.n.. (AA!); same loc., 21 Jun 1890, Korzhinsky s.n.. (LE!); same loc., 1927, Melville s.n.. (АА!), near Karkaraly, 3 Jul 1937, Dmitrieva s.n.. (AA!); same loc., same loc., 17 Jul 1987, Kupriyanov s.n.. (KG!); same loc., 18 Jun 2001, Ishmuratova s.n (KG!); the shore of Pashennoe Lake, 20 Jun 1914, s. collector 2071 (LE); 70 km southeast of Karkaraly, Kent Mountains, 19 Jul 1968, Rachkova 784 (LE!); Karkaraly Mountains, Alexandrov Klyuch, 8 Jul 1993, Mikhailov s.n.. (KG!); Karkaraly Mountains, Karkaralinka River valley, 5 Aug 1989, Denisova 692 (MW 0816794!). Irtysh: Pavlodar Region [without detailed locality] (Kusnetsov and Pavlov 1958).Kokchetav: enisovka ; NaurzumMoist pine and birch forests, along the shores of forest streams and lakes, forest swamps.Flowering in Jun–Jul; fruiting in Aug–Sep.It is included in the Red Book of Kazakhstan (category II). It is protected on the territories of the “Naurzum” State Nature Reserve; “Kokshetau” State National Nature Park, “Burabay” State National Nature Park, Karkaraly State National Nature Park; Botanical nature monument “Planting of birch and pine forests near Borovskoye Lake”.D.fuchsii, that were previously incorrectly determined as Dactylorhizarussowii. Based on these incorrectly misidentified herbarium specimens, D.russowii was previously reported for the Kokchetav FR  and in the overall flora of Kazakhstan  Soó391C9374-A15B-5AC4-B581-63BD8AED381DRussia , Kazakhstan .Kostanay Region: Auliekol District: Aman-Karagay pine forest, 1930, Dmitrieva s.n.. (AA!); Naurzum District: Naurzum-Karagai Mountains, 20 May 1909, Savich and Kucherovskaya 647 (TK!); Naurzum Reserve, the shore of Small Aksuat Lake, 2 Jul 1949, Ivleva and Kleshchina s.n. (TK!); 20 km east of Aksuat, in the upper reaches of Akbulak Stream, 12 Jun 1936, Voronov 157 (MW 0816813!); Moss swamp amongst springs on the slope of the Ulkendamdy River Valley, 23 Jun 1909, Kucherovskaya 1125 (LE!); Fedorov District: Between the Traktov and Nazaryev, on the edge of the lake basin , 7 Jul 1913, Korotkiy and Lebedeva s.n.. (LE!). Eastern Upland: Pavlodar Region: Bayanaul District: “Bayanaul” State National Nature Park, Bayanaul Mountains, 27 May 2007, Kupriyanov et al. s.n.. (KUZ 11467!). North Kazakhstan Region: Gabit Musrepov District: near the “Ardager” recreation centre, 27 Jun 2019, Kubentayev s.n.. (NUR!); the City of Petropavlovsk, near Pestroye Lake, 10 Jun 1962, Troinikova 1336 (MW 0816799!); Akkain District: west of the Borki Village, 29 Jun 1979, Kolodchenko s.n.. (NKU!). Karaganda Region: Aktogay District: the southern tip of the Kyzyl-Rai mountain system, the floodplain of the drying Karatal River, 2 Jul 1969, Mishchenkova 360 (LE!); same loc., 14 Jul 1974, Denisova 57 (LE!); same loc., in the Zhenishke River Valley, 27 Jul 1959, Denisova 224 (LE!); Bektau ata Mountains, Mikhailov and Alibekov s.n.. (KG!). East Kazakhstan Region: Abay District: the Sherubai Valley, Nura River on the shore of the Topar Reservoir, Jun 2006, Kupriyanov s.n.. (KG!); Degelen Mountains, along the shore of the Uzynbulak Stream, 29 May 1910, Kucherovskaya 309 (LE!); near the Ak-Jaman Mountains (Zhamantas), 4 Jun 1914, Shipchinsky 572 (LE!); same loc., 21 Jun 1984, Grubov et al., 603 (LE!); Chingiz Mountains, Kopa River Valley, 30 May 1914, Kosinskiy 558 (LE!); the valley of the Chagan River located 1.5 km above the mouth of the Saryzhal River, 14 Jul 1984, Korobkov 603 (LE!); Chingiztau Mountains upper Bakanas, the sources of the Kyzyluzen on the Barshatas-Abai road, 11 Jun 1984, Grubov 316 (LE!); Chingiztau Mountains, Bakanas Valley, near Ramadan Village, 9 Jun 1984, Korobkov 221 (LE!); valley of Namaz River, 28 May 1914, Kossinsky 485 (LE!); Chingiz Mountains, Munar River Valley, 27 May 1914, Shipchinsky 345 (LE!); Abraly District: valley between the Zhaksyabraly and Zhamanabraly Mountains, 4 Jun 1910, Kucherovskaya 668a (LE!); Semipalatinsk District: the western part of the mountain (Kokon) Kokentau, 15 Jun 1928, Blumenthal and Zapryagaev 475 (LE!). Kokchetav: Akmola Region: Zerendi District: near Kostomarovka, 4 Jun 1986, Mikhailov s.n.. (KG!); Archaly is 5 km from Lesogorskoe Village, 24 Jun 1929, Grigoriev s.n.. (AA!); “Kokshetau” State National Nature Park, Ormandy Bulak forestry, near Ermakovka, 28 May 2020, Kubentayev s.n.. (NUR!); Burabay District: near Dorofeevka (Akylbay), 3 Jun 1918, Drobov 305 (LE); to the SE from Shchuchya station, 28 Jun 1997, Gordyagin 20 (LE!); Burabay, Rashit - cordon, along the shore of Arykpay River, 9 Jun 2011, Khrustaleva s.n.. (KUZ 00969!); “Burabay” State National Nature Park, 101 block of Borovskoy forestry, 22 Jun 2012, Khrustaleva and Artemova 02776 (KUZ!); Zolotoborsky forestry, near Tas-Shalkar Lake, 23 Jun 2012, Khrustaleva and Artemova s.n.. (KUZ 02810!). SYRT: West Kazakhstan Region: Terekti District: near Podstepny to the southwest of Uralsk, 1895, Kulyasov 51 (MW 296063!); near Temirbek, 13 Jun 1908, Borodin et al. s.n.. (LE!). Mugodzhary: Aktobe Region: Mugalzhar District: the southern tip of the Mugodzhar Mountains, west of Algabas railway station, 21-22 Jun 1987, Skvortsov s.n.. (MHA!); horse farm Emba, 1934, Buyanov s.n.. (MHA!); Near the north-west of the Mugodzhar Mountains, in a meadow near the sands of Urkach, Dubinskaya 84 (LE!); Bol’shoy Boktybay Mountains, Berchogur place, 8 Jul 1927, Rusanov 372 (LE!); The upper reaches of the Chili River (Or) near of the place Dzhaksy-Urkach, 11 Jul 1927, Krasheninnikov 847 (LE!); To the NW from Berchogur Station on the way to Mugojar, 25 Jun 1925, Krasheninnikov 576 (LE!); at the foot of Dau-Tau Mountain, 14 Jun 1936, Khomutova and Daeva s.n.. (MW 0816798!); near the Ayryuk Mountain, 3 Jul 1927, Krasheninnikov 757 (LE!). Aktyubinsk: Aktobe Region: Martuk District: 7 km from Martuk, 17 Jun 1993, Panina s.n. (PPIU); Khobdinsky District: near Bestau Mount, 23 Jul 1934, Semsel 159 (MW 0816811!). Ulutau: Karaganda Region: Ulytau District: near Ulytau Mountains, 1842, Schrenk s.n.. (LE!); same loc., 2 Jun 2016, Nashenova and Ivanov s.n.. (ZhBG!); Arganaty Mountains, 27 Jun 2017, Kupriyanov and Khrustaleva s.n.. (KUZ 08448!); same loc., floodplain of the River Bazoy, 27 Jun 2017, Kupriyanov and Khrustaleva s.n.. (KUZ 08449!). Karkaraly: Karaganda Region: Karkaraly District: on the salty meadows near the Big Lake, 23 Jul 1890, Korzhinskiy s.n.. (LE!); Karkaraly Mountains 14 km south of Karkaralinsk, 14 Jun 1959, Denisova s.n.. (LE!); Karkaraly Mountains on the road between Karkaraly and Zharly, 22 Jun 1991, Pimenov and Klyukov 29 (MW 0816812!); near Karkaralinsk, near Zhyrym River, 12 May 1914, Kucherovskaya 1697 (LE!); near Kent Village, Kent Mountains, 2 Jun 2007, Kupriyanov et al. s.n.. (KUZ 11468!); same loc., 16 Jun 2001, Ishmuratova s.n. (KG!). Western Upland: Karaganda Region: Abay District: the shore of Talda River, near Amanzholov Farm, 20 Jun 1993, Mikhailov s.n.. (KG!); Akbastau River Valley, 3 May 2015, Alibekov s.n.. (KG!); the valley of the Sherubai, Nura River at the shore of the Topar Reservoir, Jun 2006, Kupriyanov s.n.. (KG!); Shetskiy District: Shopa River Valley, foothills Kusmuryn, 25 Jun 1994, Mikhailov s.n.. (KG!).Tobol-Ishim: Wet meadows, flooded saline meadows, valleys of rivers, streams, lakeshores.Flowering in Jun–Jul; fruiting in Aug–Sep.Not protected.Dactylorhizaincarnata is the most common orchid species in the studied region, found in almost all areas. However, for Turgay FR, it was recently reported by mistake. The corresponding localities (FR. There are currently no known orchid species that occur in Turgay FR. Existing reports of D.traunsteineri (Saut. ex Rchb.) Soó for Kazakhstan are most likely based on misidentified D.incarnata. Therefore, the report of D.traunsteineri for Urkach Plateaeu in Mugodzhary (Kusnetsov and Pavlov 1958) is possibly based on a herbarium specimen in LE with incomplete label , which was probably collected near the end of May 1842 ). In our opinion, this specimen corresponds to Dactylorhizaochroleuca. The identification is based on information provided on the herbarium label . However, it is possible that these plants belong to hypochromic variants of D.incarnata. Exact determination is possible only through allozyme analysis  Sоó4B386E95-6604-5B93-9DBB-498D5375892BRussia , Kazakhstan (?Altai).Akmola Region: Burabay District: “Burabay” State National Nature Park: the shore of Svetloye Lake, 8 Jun 1960, Denisova 1326 (MW 0816814!); Barmashinsky forestry, 7 Jun 2019, Kubentayev s.n.. (NUR!); same loc., the planning quarter 134, 18 Jun 2012, Artemova (KUZ 02650!); the swampy shore of Karas’e Lake, Khrustaleva and Artemova s.n.. (KUZ 02696!). Karkaraly: Karaganda Region: Karkaraly District: Karkaraly Mountains, 12 Aug 2006, Kupriyanov and Manakov s.n.. (KUZ 11465!). Mugodzhary: Aktobe Region: Mugalzhar District: Mugodzhary Mts, “Urkach” place  Soó437D2305-3B48-52A1-A053-27885585CD24Russia (Siberia), Kazakhstan .Karaganda Region: Aktogay District: near Aktogay, 24 Jun 1917, Harin s.n.. (AA!). Western Upland: Karaganda Region  ; near Naurzum-Karagai, 20 May 1909, Savich and Kucherovskaya s.n.. (LE!); near Karamenda, the shore of Sarymoyin Lake, 1 Jul 1911, Borodin s.n.. (LE!); Egin-Bulak spring, north of Naurzum Forest, 22 Jun 1934, Pavlov 1396 (MW 0816830!); 5 km south of Aksuat Village, 20 Jun 1945, Voronov s.n.. (MW 0816882!).Eastern Upland: ocality) . Tobol-ILowlands amongst birch trees, damp saline meadows and floodplains of rivers.Flowering in Jun–Jul; fruiting in Aug–Sep.Not protected.Dactylorhizasalina is reported here for the first time for the Tobol-Ishim FR and Kostanay Region. D.salina was recently erroneously reported for the Turgay FR , Kazakhstan (Altai).Pavlodar Region: Bayanaul District: Bayanaul Mountain Forest, on the slope of the watershed between Jasybai Lake and Sabyndykul, 24 Jun 1979, Lalayan s.n (SVER 627698!). East Kazakhstan Region: the village of Kriushi, meadow along a stream, 21 Jul 1928, Ilyin and Heinrichson s.n.. (LE!).Eastern Upland: Stream valleys, swampy meadows.Flowering in Jun–Jul; fruiting in Aug–Sep.Not protected.Dactylorhizasibirica is reported for the studied region for the first time. This allopolyploid species was described relatively recently, in 2016, with diploid Siberian D.fuchsii and D.incarnata as its presumable parental taxa  Nevski or Dactylorhiza×kerneri  Nevski4473B460-9C46-5BAC-B4C2-A324CD8902F9Russia (Siberia), Kazakhstan .Akmola Region: Burabay District: near the Mirnaya Dolina cordon, the shore of Karabulak Stream, 25 Jun 1937, Sobolev s.n.. (AA!). Tobol-Ishim: Kostanay Region: Naurzum District: Nauryzym-Karagay Mountains, 20 May 1909, Savich and Kucherovskaya 649 (LE!); “Naurzum” State Nature Reserve, 4 Jun 1938, Siu s.n.. (MW 0816881!). Mugodzhary: Aktobe Region: Shalkar District: near Ber-Chogur, 10 Jun 1927, Rusanov s.n. (AA!); northwest of Ber-Chogur, on the road to Mugodzharsk, 25 Jun 1927, Krasheninnikov s.n.. (AA!). Western Upland: Karaganda Region: Zhanaarka District: Sarysu River Valley, “Kara-Agach” place, 13 Jun 1949, Goloskokov s.n.. (AA!). Eastern Upland: East Kazakhstan Region: Ayagoz District: Chingizstau, upper reaches of Kalguta River, 17 Jun 1958, Gamayunov s.n.. (AA!).Kokchetav: Valleys of rivers and streams, along the damp edges of birch and aspen forests, through swamps, salt marshes, wet meadows, in the lowlands amongst birch thickets.Flowering in Jun–Jul; fruiting in Aug–Sep.Not protected.D.majalis. for Mugodzhary (near Ber-Chogur and Mount Boktybai). Although corresponding herbarium specimens were not located, we believe that this report is an obvious mistake. It is more likely that the plants were D.umbrosa, as we found herbarium materials collected from the same place ). We consider D.umbrosa and D.salina to be closely-related species and determining plants with certainty can sometimes be challenging.Taxon classificationPlantaeAsparagalesOrchidaceae(Hoffm.) Besser54835AAF-8718-53BD-8D69-DC3A94AD59E8Russia .Kostanay Region: Uzynkol’skiy District: near Krasnye Borki, 12 Jul 1990, KSPI students 2189 .Tobol-Ishim: Pine forestsFlowering in Jun–Jul; fruiting in Aug–Sep.Not protected. The species is very rare and we recommend to include it in the next edition of the Red Book of Kazakhstan.For the flora of Kazakhstan, the species was reported relatively recently by Taxon classificationPlantaeAsparagalesOrchidaceae(L.) Crantz74F2BC94-2086-5998-97C0-E9703107679ERussia , Kazakhstan .Aktobe Region: ?Mugalzhar District: Mugodzhar Mts [without detailed locality]  Crantz857D060E-3F03-5086-9497-8C87CFF47B71Russia , Kazakhstan .Aktobe Region: Uilskiy District: near Uil, 21 Aug 1936, Nikitin & Deulina s.n. (LE!). Mugodzhary: Aktobe Region: Mugalzhar District: Along the shore of the Shuldak River, 22 Jun 1927, Rusanov s.n.. ; Akzerendy River Valley, 4 Jul 1927, Rusanov s.n.. (AA! LE!); Mugodzhary Mts, the Kunduzdy River Valley (left tributary of the Emba), 20 Jul 1857, Borszczov 299 (LE!); Shalkar District: Shuldak River Valley (Shet-Irgiz), 29 Jun 1927, Krashenninikov 638 (LE!). SYRT: West Kazakhstan Region: Chingirlauskiy District: “Kara-Agach” place, 13 Jun 1950, Nikishin s.n.. (LE!). Tobol-Ishim: Kostanay Region: Auliekol District: Aman-Karagay pine forest, 10 Sep 1921, Pavlov s.n.. (LE!); same loc., near Novonezhinka, 3 Jun 1925, Rusanov s.n.. (LE!); Mendykara District: Borovskaya water protection forest dacha, near Borovskoye, 08 Jul 1925, Rusanov s.n.. (LE!); Nauruzymsky District: Ak-Kuchuk River Valley, 1 Aug 1929, Vernander s.n.. (LE!); Kokchetav: Akmola Region: Burabay District: Northern swampy shore of Shchuchy Lake, 3 Sep 1981, Gorchakovskiy s.n.. (SVER 715630!). Irtysh: Pavlodar Region: Akkuli District: the shore of Yamyshevskoye Lake, 26 Jun 1913, Kucherevskaya 1048 (LE!). Semipalatinsk Pinery: East Kazakhstan Region: Semipalatinsk District: 25 km east of Semipalatinsk, Kashtak place, 10 Jul 1933, Sumnevich s.n.. (TK!); same loc., 20 Aug 1933, Sumnevich s.n.. (TK!) near the Semeytau Mountains, Northern Spring, 25 Jun 1914, Mordvinova s.n.. (MOSP!); Borodulikha District: Semeytau Mountains, near the farm, 6 Aug 1928, Zapryagaev 1973 (LE!); Beskaragaysky District: near the mouth of the Shagan River, 23 Sep 1928, Zapryagaev 2452 (LE!). Karkaraly: Karaganda Region: Karkaraly District: Zheltau Mountains . It is protected in the following territories: “Naurzum” State Nature Reserve, “Semey Ormany” State Nature Reserve, “Burabay” State National Nature Park, “Karkaraly” State National Nature Park, as well as the State Nature Reserves of “Floodplain of the Irtysh River”, “Orkash”, “Kokzhide-Kumzhargan” and “Budarinskiy”. It is also protected in the natural monument “Birch and pine plantations forests near Borovskoye Lake”. Taxon classificationPlantaeAsparagalesOrchidaceaeSw.5EF385DA-958B-5D55-8961-647F653825F9Russia , Kazakhstan (Altai).Karaganda Region: Karkaraly District: Kent Mountains, “Karaagash” place, 4 Aug 1986, Kupriyanov and Mikhailov s.n.. (KG!).Karkaraly: Swampy pine forests, swamps.Flowering in Jul–Aug; fruiting in Sep–Oct.This species is included in the Red Book of Kazakhstan (category II) as a rare species found in small numbers within a limited area. In the studied region, it is preserved in the “Karkaraly Biological Reserve”.The species in the studied region is known from a single locality in central Kazakhstan, as confirmed by the above herbarium sample. This finding was published in 1987 . CurrentTaxon classificationPlantaeAsparagalesOrchidaceae(L.) R.Br.66297488-7408-5EF8-B086-20DA7086C272Russia , Kazakhstan .Akmola Region: Burabay District: near Karas’e Lake, 19 Jul 1913, Semenov s.n.. (TK!); same loc., 10 Aug 1973, Gorchakovskiy s.n.. (SVER 695775!); same loc., 12 Jun 2011, Kupriyanov s.n.. (KUZ 00893!); same loc., small southern swamp, 26 May 1973, Gorchakovskiy s.n.. (SVER 695772!); same loc., big swamp, 29 Jun 1974, Gorchakovskiy s.n.. (SVER 695770!); Sinyukha Mountain, 17 Jun 1912, Semenov s.n.. (TK!); same loc., north slope, 24 Jun 1929, Ilyin s.n.. (LE!); Zolotoborskiy forestry, sq. 24, 18 Jun 1972, Gorchakovskiy s.n.. (SVER 695773!); Borovsky forest area, near Svetloe Lake, 9 Aug 1973, Gorchakovskiy s.n.. (SVER 695774!); same loc., near Shortankulskiy peat bog, 5 Aug 1978, Gorchakovskiy s.n.. (SVER 695776!); Kokchetav Upland, upper reaches of the Imanayevsky Spring, 2 Jul [presumably 1895-1910], Gordiagin 593 (LE); near the top of Ush-Tas Mount, 2 Jul 1896, Gordyagin 1105 (LE!); near Burabay, “Burabay” State National Nature Park, Barmashinskoe forestry, 16 Jul 2019, Kubentayev s.n.. (NUR!).Kokchetav: Moist pine, birch and mixed forests.Flowering in Jul–Aug; fruiting in Sep–Oct.G.repens in this area are exposed to strong recreational influences.Not protected. It is necessary to strengthen security measures in the territory of the Shchuchinsko-Borovskaya resort area in the Kokchetav Upland as the habitats of Goodyerarepens  R.Br.AE002BE5-FC77-548C-B28D-5FFD545A7801Russia , Kazakhstan (Altai).Akmola Region: Burabay District: Kokchetav Upland, near Burabay, “Burabay” State National Nature Park, Barmashinskiy forestry, 12 Jul 2019, Kubentaev s.n.. (NUR!); Kokchetav Upland, at the top of Mount Sinyukha, 22 Jul 1960, Denisova 1709 (MW 0816897!); Bulandynskiy District: Otradnenskiy forestry, forest quarter no. 121, 4 Aug 1960, Denisova 2069 (MW 0816896!), same loc., forest quarter no. 11, 4 Aug 1960, Denisova 1270 (MW 0816895!); Aryk-Balykskiy District: Kokchetav Upland, hills to the south of Imantau Lake, 2 Aug 1960, Denisova 1817 (MW 0816898!); Zerendi District: 30 km south of Kokshetau, 7 Jul 1957, Borisova & Rachkovskaya 345 (LE!). Tobol-Ishim: Kostanay Region: Auliekol District (Auliekol’skiy District): near Aman-Karagay, 27 Apr 1929, Vernander et al. 331 (LE!). North Kazakhstan Region: Kyzylzhar District: right shore of Ishim River, near Krasnoyarka, 29 Jun 1971, Sologub and Zelinskaya s.n.. (NKU!); same loc., 29 Jun 1971, Stepanova and Shahvatova s.n.. (NKU!); same loc., 3 Jul 1971, Troskina and Shandybina s.n. (NKU!); same loc., Aug 1973, Wenzler et al. s.n.. (NKU!); same loc., 6 Jul 1974, Shushakova and Konovalov s.n.. (NKU!); same loc., 06 Jul 1974, Gorbunova and Mikheeva s.n.. (NKU!); same loc., 9 Jul 1974, Sinichkina et al. s.n. (NKU!); same loc., 13 Jul 1974, Fomenko s.n.. (NKU!); same loc., 14 Jul 1974, Sokolovskaya s.n.. (NKU!); same loc., 14 Jul 1974, Spirenkova and Temirbaeva s.n.. (NKU!). Irtysh: Pavlodar Region: Shcharbakty District: near Aleksandrovka, 1885, Golde s.n. (LE!). Semipalatinsk Pinery: East Kazakhstan Region [without detailed locality] (Kusnetsov and Pavlov 1958).Kokchetav: Meadows, birch spikes and grassy pine forests.Flowering in Jun–Jul; fruiting in Aug–Sept.Not protected. We recommend to include this species in the next edition of the Red Book of Kazakhstan.FR) and Kokchetavskiy Uezd (belongs to Kokchetav FR).The species was seriously under-recorded in earlier treatments, mainly due to incorrect determinations of the existing herbarium specimens. Thus, no reports are available for Kokchetav and Tobol-Ishim FRs, Kostanay, North Kazakhstan and Akmola administrative regions in the floristic accounts of Kazakhstan (Kusnetsov and Pavlov 1958), Kazakh Upland  and TurgTaxon classificationPlantaeAsparagalesOrchidaceae(L.) KuntzeC52725B2-9122-5B11-986C-8E9B6E008FB7Russia .Aktobe Region: Mugalzhar District: Mugodzhary Mts, “Urkach” place, 1 Sep 1934, Samseev 514 (MW 0816996!).Mugodzhary: Sphagnum swamps.Flowering in Jun–Jul; fruiting in Aug–Sep.Not protected. We recommend to include this species in the next edition of the Red Book of Kazakhstan.Hammarbyapaludosa was only recently reported for Kazakhstan for the first time (Microstylismonophyllos (L.) Lindl. This location is the southernmost part of the area of H.paludosa. Fieldwork is necessary to check whether H.paludosa is still extant in that locality.rst time . It was Taxon classificationPlantaeAsparagalesOrchidaceae(L.) Y.Tang, H.Peng & T.Yukawa14FD558B-60F6-535A-AC72-353B9FA18E75Neottianthecucullata (L.) Schltr. ≡Ponerorchiscucullata (L.) X.H.Jin, Schuit. & W.T.Jin.  ≡Russia .Akmola Region: Burabay District: 3 km north of Shuchinsk, 1978, Grudzinskaya s.n.. ; Borovskoy forest area, near Shortankulskiy peat bog, 5 Aug 1978, Gorchakovskiy s.n.. (SVER!); near Balkashino, in the northern part of the forest dacha of B. Tyukty, 9 Aug 1929, Grigoriev 367 (LE!); the northern slope of Tuyak-Tau Mountain, 13 Jul 1901, Gordyagin 529 (LE!); near the Ush-Tas Mount top, 2 Jul 1896, Gordyagin 1105 (LE!); the eastern shore of Svetloe Lake, 16 Jul 2019, Kubentaev and Alibekov s.n.. (NUR!); near Burabay, “Burabay” State National Nature Park, Akylbayskoye forestry, 16 Jul 2019, Kubentayev s.n.. (NUR!); same loc., Borovskoe forestry, 16 Jul 2019, Kubentayev s.n.. (NUR!); Zerendi District: “Kokshetau” State National Nature Park: Jilandinskoe forestry, near the “Gorodok” cordon, 11 Aug 1960, Denisova 1973 (MW 0816899!); Ayrtau District: near Shokkaragai, 10 Aug 2020, Kubentaev et.al. s.n.. (NUR!). Eastern Upland: Pavlodar Region: Bayanaul District: Bayanaul Mountains .Kokchetav: ountains ; same loPine and birch forests.Flowering in Jul–Aug; fruiting in Sep–Oct.H.cucullata  R.Br.7AA7A4D8-9CBC-52C6-9694-659910CADA4BRussia , Kazakhstan (Altai).East Kazakhstan Region: Semipalatinsk District: near Semipalatinsk, 8 Jun 1914, Mordvinova s.n.. (MOSP!). Irtysh: Pavlodar Region [without detailed locality]  Rich.8158186E-6194-500F-867E-74E5FBD3459DRussia . Kazakhstan .Akmola Region: Burabay District: near Borovoye, the shore of Chebach’ye Lake, 12 Jun 1913, Drobov 431 (LE!). Semipalatinsk Pinery: East Kazakhstan Region: Semipalatinsk District: near Semipalatinsk, 8 Jun 1914, Mordvinova s.n.. (MOSP!). Mugodzhary: Aktobe Region: Mugalzhar District: Mugodzhary Mts, “Urkach” place, 21 Aug 1927, Krasheninnikov 1230 (LE!).Kokchetav: Sedge and sphagnum swamps.Flowering in Jun–Jul; fruiting in Aug–Sept.Not protected. The species is extremely rare and we consider it mandatory to include it in the next edition of the Red Book of Kazakhstan.Liparisloeselii is reported here for the first time for Semipalatinsk Pinery and Mugodzhary FRs. Previously, in the studied region, the species was observed only in Kokchetav FR  Sw.78AF6877-8E9C-5F95-907D-A353C6E4DE7ERussia .Kostanay Region: Mendykara District: Borovskaya water protection forest dacha, 17 Jul 1925, Rusanov 1553 (LE!); near Borovskoye ; same loc., 10 km west of Bayan-Aul, 19 Jul 1959, Denisova 436 (LE!); same loc., 19 Jul 1959, Denisova 427 (MW 0816997!). Karkaraly: Karaganda Region: Karkaraly District: Zhisil’tau Mountains, near Egindibulak, 19 Jul 1992, Kupriyanov s.n.. (KG!).Tobol-Ishim: rovskoye ; near Karovskoye ; Auliekorovskoye . EasternGrassy birch forests, near lakes, along forest streams.Flowering in Jun–Jul; fruiting in Aug–Sept.Not protected. We recommend to include this species in the next edition of the Red Book of Kazakhstan.M.monophyllosvar.diphyllos (Cham.) Luer  Rchb.f.1041F267-76DC-5B88-A732-0AE63E6AE325Russia (Siberia), Kazakhstan .Karaganda Region: Karkaraly District: Kent Mountains, near Kent, 1 Jun 2007, Kupriyanov et al. (KUZ 11470!); same loc., “Auletas” place, 25 Jul 1992, Kupriyanov s.n.. (KG!); Zhisil’tau Mountains, near Egindibulak, 16 Jul 1992, Mikhailov s.n.. (KG!). Eastern Upland: Pavlodar Region: Bayanaul District: Bayanaul Mountains, 10 km west of Bayan-Aul, 19 Jul 1959, Denisova 444 (MW 0816977!); same loc., 23 Jul 1963, Denisova 1495 (MW 0816976!).Karkaraly: Damp birch-aspen forests along the shores of streams and lakes.Flowering in Jun–Jul; fruiting in Aug–Sep.Not protected. The species is extremely rare and we consider it mandatory to include it in the next edition of the Red Book of Kazakhstan.Neottiacamtschatea is reported here for the first time for Eastern Upland FR and for Pavlodar Region. Earlier occurrences of the species were confirmed in eastern Kazakhstan , southern Kazakhstan  (Kusnetsov and Pavlov 1958) and central Kazakhstan   .Taxon classificationPlantaeAsparagalesOrchidaceaeL.CC7FA3AF-1CAE-5881-9AF3-929F5A7D8DFBRussia .West Kazakhstan Region: Chingirlauskiy District: “Kara-Agach”, 23 Jun 1950, Nikitin s.n.. (LE!); same loc., place valley of the Ural River, headwaters Buldurta River, 21 Jun 2003, Darbaeva s.n.. (LE!). EMBA: Aktobe Region: Mugalzhar District: near Emba, 30 May 1840, Bunge 1334 (LE!). Mugodzhary: Aktobe Region: Mugodzhary Mts “Urkach” place, near Kumyskul Lake, 10 Jul 1927, Rusanov 773 (LE!), s.n.. (AA!); Mugodzhary Mts, near Ayrik ; Semipalatinsk Pinery: East Kazakhstan Region: Semipalatinsk District: near Semipalatinsk, 8 Jun 1914, Mordvinova s.n.. (MOSP!); Beskaragajskiy District: near Kanonerka, 12 Jun 1996, Kupriyanov et al. s.n.. (ALTB!). Eastern Upland: East Kazakhstan Region: Abaj District: Akshatau Ridge . It is protected in the territory of the following State Nature Reserves: “Semey Ormany”, “Orkash”, “Kokzhide-Kumzhargan”, “Budarinsky”, “Kirsanovsky” and “Ak-Kuma”. In Kazakhstan, Taxon classificationPlantaeAsparagalesOrchidaceae(L.) Rich.1471CFF5-72B2-55F7-80FE-48B87441F8D8Russia , Kazakhstan (Altai).Kostanay Region: Mendykara District: Borovskaya water protection forest dacha, 28 Jul 1923, Rusanov s.n.. (LE!); near Borovskoye, 10 Jul 1977, Pugachev s.n.. (LE!); botanical nature monument “Plantations of birch and pine forests near Borovskoye Lake”, 24 Jun 2009, students 195 (KSPI!); Zhitikara District: botanical nature monument “Relict larch-birch grove with Sukachev larch”, 10 Jun 2012, Perezhogin ; Auliekol District: near Kazanbasskoye ; same loc., 29 Jun 1971, Stepanova and Shakhvatova s.n.. (NKU!); same loc., 29 Jun 1971, Sologub and Zelinskaya s.n.. (NKU!); same loc., 28 Jun 1971, Fesan and Kosmagambetova s.n.. (NKU!); same loc., 29 Jun 1971, Trushcheleva and Shirokikh s.n.. (NKU!); same loc., 26 Jun 1972, Kudinova and Schneider s.n.. (NKU!); same loc., 24 Jun 1972, Makayun s.n.. (NKU!); same loc., 27 Jun 1972, Rosinskaya and Khrushchev s.n.. (NKU!); same loc., 29 Jun 1972, Shegebaev and Zhampeisov s.n.. (NKU!). Kokchetav: North Kazakhstan Region: Aiyrtau District: near Lobanovo, Kozhevnya swamp, 28 May 2020, Kubentaev et al. s.n.. (NUR!).Tobol-Ishim: basskoye ; near Kabasskoye ; near Aubasskoye . North KForest edges and glades, dry meadows, scrub thickets, thinned forests and the outskirts of bogs.Flowering in May–Jul; fruiting in Jul–Aug.The species is included in the Red Book of Kazakhstan (category III) as an endangered species. It is protected on the territory of the nature monument “Stands of birch and pine forests near Borovskoye Lake”; the botanical nature monument “Relict larch-birch grove with Sukachev larch”; Kokshetau State National Nature Park; and the “Sogrov” State Nature Reserve.P.bifolia for Kokchetav FR  by There is an old report of Taxon classificationPlantaeAsparagalesOrchidaceae(R.Br.) Lindl64D92CF3-B80F-5524-99B4-A67238C876A5Russia , Kazakhstan (Altai).Kostanay Region: Mendykara District: 4 km south of Borovskoye, 20 Jun 1925, Rusanov s.n.. (LE!); same loc., 19 Jun 1925, Rusanov 1490 (MW 0816970!); Auliekol District: near Kalininskoye . Irtysh: Pavlodar Region [without detailed locality] (Kusnetsov and Pavlov 1958).Tobol-Ishim: ninskoye . KokchetOn peat bogs.Flowering in Jul–Aug; fruiting in Aug–Sept.Not protected. Taking into account the limited expansion and the small number of individuals in the population, we recommend to include this species in the next edition of the Red Book of Kazakhstan.Spiranthesaustralis in the study region, since the species was not observed here for almost 30 years.Currently, field studies are needed to verify the presence of