Distinct Taxa Research Articles

Medieval fish remains on the Newport ship identified by ZooMS collagen peptide mass fingerprinting

Fish represent a key economic, social and ecological group of species that humans have exploited for tens of thousands of years. However, as many fish stocks are going into decline and with little known about the anthropogenic impacts on the health of the marine ecosystem pre-Industrial Revolution, understanding historical and archaeological exploitation of fish species is key to accurately modelling these changes. Here, we explore the potential of collagen peptide mass fingerprinting (also known as Zooarchaeology by Mass Spectrometry, or ZooMS) for identifying fish remains from the Medieval (fifteenth century) Newport ship wreck (Wales, UK), and in doing so we establish a set of biomarkers we consider useful in discriminating between European fish taxa through the inclusion of over 50 reference taxa. The archaeological results identified nine distinct taxonomic groups, dominated by ling (> 40%), and a substantial amount of cod (> 20%) and hake (~ 20%). The vast majority of samples (> 70%) were identified to species level, and the inability to identify the remaining taxonomic groups with confidence using ZooMS was due to the fact that the reference collection, despite being relatively large in comparison to those presented in mammalian studies, reflects only a small proportion of fish biodiversity from this region. Although the results clearly demonstrate the potential for ZooMS as a means of fish bone identification, the sheer number of different fish species that potentially make up ichthyoarchaeological assemblages leads to obvious requirements for the analysis on much greater numbers of modern reference specimens, or the acquisition of collagen sequences.

Integrative taxonomy reveals overlooked cryptic diversity in the conifer feeding Batrachedra pinicolella (Zeller, 1839) (Lepidoptera, Batrachedridae)

During efforts to generate DNA barcodes for all European Lepidoptera, Batrachedrapinicolella (Zeller, 1839) was found to comprise two genetically distinct clusters. Morphological investigation and results from two nuclear markers and ddRAD sequencing furthermore support the existence of two distinct taxa which we treat as two separate species, B.pinicolella and B.confusellasp. nov. A lectotype for B.pinicolella is designated. Available data indicate that the biology of both species also differs, with Piceaabies (L.) Karsten as a proved host-plant for B.pinicolella and Pinussylvestris L. for B.confusellasp. nov. Both species are mainly distributed on the European continent with B.pinicolella occurring in boreal parts of North and Central Europe and introduced to Canada, reflecting a boreo-montane distribution pattern. Batrachedraconfusellasp. nov. is more widely distributed in temperate Northern and Central Europe.

Multi-Gene Phylogeny of the Ciliate Genus Trachelostyla (Ciliophora, Hypotrichia), With Integrative Description of Two Species, Trachelostyla multinucleata Spec. nov. and T. pediculiformis (Cohn, 1866).

Many hypotrich genera, including Trachelostyla, are taxonomically challenging and in a need of integrative revision. Using morphological data, molecular phylogenetic analyses, and internal transcribed spacer 2 (ITS2) secondary structures, we attempt to cast more light on species relationships within the genus Trachelostyla. The present multifaceted approach reveals that (1) a large-sized species with numerous macronuclear nodules, isolated from sandy littoral sediments in southern China, is new to science and is endowed here with a name, T. multinucleata spec. nov.; (2) two other Chinese populations previously identified as T. pediculiformis represent undescribed species; and (3) multigene phylogeny is more robust than single-gene trees, recovering the monophyly of the genus Trachelostyla with high bootstrap frequency. Additionally, ITS2 secondary structures and the presence of compensatory base changes in helices A and B indicate the presence of four distinct taxa within the molecularly studied members of the genus Trachelostyla. Molecular data are more suitable for delimitation of Trachelostyla species than morphological characters as interspecific pairwise genetic distances of small subunit (18S) rDNA, ITS1-5.8S-ITS2, and large subunit (28S) rDNA sequences do not overlap, whereas ranges of multiple morphometric features might transcend species boundaries.

Scyliorhinus hachijoensis, a new species of catshark from the Izu Islands, Japan (Carcharhiniformes: Scyliorhinidae).

A new species of catshark genus Scyliorhinus, S. hachijoensis sp. nov., is described for the islands of Mikurajima, Hachijojima, and Torishima in southeastern Japan. Scyliorhinus hachijoensis has clasper hooks, which is a common feature in males of the most closely related species (S. torazame), but is distinguished by its coloration (presence of dark spots), the height of its anal fin (higher than the caudal peduncle), and the shape of pectoral and pelvic fins, and dermal denticles. Molecular data also corroborates the new species as a distinct and monophyletic taxon by nucleotide sequence analysis of three mitochondrial DNA regions.

White-footed dunnarts (

A population of white-footed dunnarts (Sminthopsis leucopus) occurs in the Wet Tropics bioregion of tropical north Queensland, Australia separated by about 1800 km from conspecifics in temperate New South Wales, Victoria and Tasmania. We conducted targeted surveys for S. leucopus in north-east Queensland and obtained new records, including the first reported capture of the species in Queensland in 18 years. We assessed the genetic and morphological divergence of the north Queensland population against New South Wales, Victorian and Tasmanian S. leucopus, in conjunction with distribution and habitat differences, to assess whether this isolate should be described as a distinct taxon. Sequencing of the mitochondrial Cytochrome b gene revealed genetic divergence estimates of 2.3–2.8% and 4.3–4.8% between the north Queensland population and S. l. ferruginifrons (Victoria) and S. l. leucopus (Tasmania) respectively. Based on genetic divergence, cranial morphology, differences in habitat, and geographical isolation, we describe the north Queensland population as a new subspecies of Sminthopsis leucopus. We suggest a conservation classification of Endangered given its small distribution, apparent low density, tropical upland location and potential threats, especially related to climate change.

Taxonomic entity of Saussurea taquetii (Asteraceae) compared with S. japonica and S. pulchella

Saussurea taquetii reported from Jejudo Island is either treated as a synonym of S. japonica or is recognized as a distinct taxon. Saussurea japonica and S. pulchella belong to the sect. Theodorea by having peculiar outer phyllaries with pale purplish scarious apical appendages; they are closely related to each other morphologically and are known to occur widely throughout the Korean Peninsula. To assess the taxonomic status of S. taquetii, we investigated representative populations of S. taquetii from jejudo Island, Korea, and S. japonica from Kyushu in Japan. We conducted a comparative study morphologically using specimens of three species from KH, CNU, and TI. Saussurea taquetii is very similar morphologically and ecologically to S. japonica in Kyushu. Unlike previous floristic treatments, we concluded that the two taxa, S. pulchella and S. taquetii, occur on the Korean Peninsula and on jejudo Island, respectively.

Rhyacophila siparantum sp. nov. (Trichoptera: Rhyacophilidae), a new species of the R. philopotamoides species group from the Republic of Kosovo with molecular and ecological notes

In this study we describe Rhyacophila siparantum sp. nov., a new species of the Rhyacophila philopotamoides species group from the Bjeshkët e Nemuna Mountains in Kosovo, based on morphological (male adults) and molecular (sequencing of the barcode region of the cytochrome c oxidase subunit I gene (COI)) characteristics. The new species is morphologically closest to Rhyacophila schmidinarica Urbanič, Krušnik & Malicky, 2000, and also closely resembling Rhyacophila hirticornis McLachlan, 1879. R. siparantum sp. nov. differs from both its most similar congeners primarily by the shape of the segment X, which is in lateral view short apically and with a pronounced lateral hump on the posterior edge. This difference, combined with other characters related to the shape of the second segment of inferior appendages, spine pattern of parameres and size of ventral teeth on segments VI and VII, make R. siparantum sp. nov. easily distinguishable from both most similar congeners. Phylogenetic and taxonomic relationships were reconstructed using two methods of phylogenetic inference, and two species delimitation methods. All this supports Rhyacophila siparantum sp. nov. as a distinct taxon. 
 The adults of Rhyacophila siparantum sp. nov. were found during the period May – August, nearby a small rheocrene spring inside a forested area. The new species is most probably a microendemic of the Bjeshkët e Nemuna, a mountainous massive known for several other endemic species of caddisflies. Rhyacophila siparantum sp. nov. is the seventeenth known species of the genus Rhyacophila Pictet, 1834 from Kosovo.

Are Palmer’s Elm-Leaf Goldenrod and the Smooth Elm-Leaf Goldenrod Real? The Angiosperms353 Kit Provides Within-Species Signal in Solidago ulmifolia s. l.

Abstract— The genus Solidago represents a taxonomically challenging group due to its sheer number of species, putative hybridization, polyploidy, and shallow genetic divergence among species. Here we use a dataset obtained exclusively from herbarium specimens to evaluate the status of Solidago ulmifolia var. palmeri, a morphologically subtle taxon potentially confined to Alabama, Arkansas, Mississippi, and Missouri. A multivariate analysis of both discrete and continuous morphological data revealed no clear distinction between S. ulmifolia var. palmeri and Solidago ulmifolia var. ulmifolia. Solidago ulmifolia var. palmeri’s status was also assessed with a phylogenomic and SNP clustering analysis of data generated with the “Angiosperms353” probe kit. Neither analysis supported Solidago ulmifolia var. palmeri as a distinct taxon, and we suggest that this name should be discarded. The status of Solidago delicatula (formerly known as Solidago ulmifolia var. microphylla) was also assessed. Both morphological and phylogenetic analyses supported the species status of S. delicatula and we suggest maintaining this species at its current rank. These results highlight the utility of the Angiosperms353 probe kit, both with herbarium tissue and at lower taxonomic levels. Indeed, this is the first study to utilize this kit to identify genetic groups within a species.

MOLECULAR CHARACTERIZATION OF ANISAKID NEMATODES HYSTEROTHYLACIUM SPECIES FROM JAPANESE THREADFIN BREAM NEMIPTERUS JAPONICUS (BLOCH, 1791) (PERCIFORMES, NEMIPERIDAE) FROM IRAQI MARINE WATER FISH

The present study provides a new insight into valuable information on the diverse structure of the Anisakid population and discusses the limited species richness in the Nemipterus japonicus (Bloch,1791) (Perciformes, Nemiperidae). The fishing area consists of various locations in the Arabian Gulf (29°58 0 33 00 N48°28 0 20 E). A total of 315 marine fish were examined, (n=287) were infected. Larval stages (n= 763) encysted within the mesenteries peritoneum and viscera of fish organs were isolated, with a prevalence of 91.11% of infection and, the intensity was 2.65. Molecular analysis was carried out on thirty individuals who have examined the morphology and showed some appearance differences, by amplifying internal transcribed spacers ITS and ITS-1 of nuclear rDNA (rDNA) by PCR using the primer sets NC5/NC2 and SS1/NC13R of thirteen DNA products. Evolutionary analyses were conducted in MEGA X. based on the identity percentage in the GenBank database showed that they belong to anisakid nematodes, in particular, they belong to eleven distinct taxa within the Hysterothylacium Ward & Magath, 1917 (Rhabditida, Raphidascarididae) and one identified species H. amoyense (Hsü, 1933) Deardorff & Overstreet, 1980. The current study records eleven species that belong to a genus of Hysterothylacium; some of the alignment of sequences polymorphisms reveals new different individuals of larvae species that may be adopted as new species if their adult stage is detected, and N. japonicus fish considered as a new host record. The current study provides some insights on the systematic taxonomy of these parasites, in addition, it supports similar studies that have been published elsewhere.

Taxonomic notes on Philippine Hornstedtia (Zingiberaceae) including a description of a new species

A new species, Hornstedtia olivacea Docot & Pranada, from the province of Quezon, Philippines is described here with information on its distribution, habitat and phenology, along with a provisional IUCN conservation assessment. A second-step lectotypification of Hornstedtia conoidea Ridl. is proposed and a previous lectotypification of Hornstedtia microcheila Ridl. is clarified. Examination of the collection Ramos & Edaño 44454 supports the occurrence of Hornstedtia havilandii (K.Schum.) K.Schum. in the Philippines but new material is needed to check if the populations in the province of Sulu are distinct enough from the populations in Borneo to be recognised as a distinct taxon. An updated key to the Hornstedtia species of the Philippines is also provided.

Conserving evolutionarily distinct species is critical to safeguard human well-being

Although there is growing interest in safeguarding the Tree of Life to preserve the human benefits that are directly provided by biodiversity, their evolutionary distribution remains unknown, which has hampered our understanding of the potential of phylodiversity indicators to evince them. Here, I drew on a global review of plant benefits and comprehensive phylogenetic information to breakdown their evolutionary distribution and thereby show why the commonly used Phylogenetic Diversity and Evolutionary Distinctiveness indicators can unequivocally help to preserve these natural services. Beneficial species clumped within phylogenetically overdispersed genera and closely related species often contributed very few and redundant benefits, suggesting that multiple plant lineages are required to maintain a wide variety of services. Yet, a reduced number of species stood out as multi-beneficial and evolutionarily distinct plants relative to both the entire phylogeny and the subset of beneficial species, and they collectively contributed a higher-than-expected number of records for most types of benefits. In addition to providing a clear mechanistic understanding for the recently proved success of Phylogenetic Diversity in capturing plant benefits, these findings stress the decisive role that conservation programmes aimed at protecting evolutionarily distinct taxa will play in safeguarding the beneficial potential of biodiversity for the future.

Testing the agreement of trees with internal labels

BackgroundA semi-labeled tree is a tree where all leaves as well as, possibly, some internal nodes are labeled with taxa. Semi-labeled trees encompass ordinary phylogenetic trees and taxonomies. Suppose we are given a collection {mathcal {P}}= {{mathcal {T}}_1, {mathcal {T}}_2, ldots , {mathcal {T}}_k} of semi-labeled trees, called input trees, over partially overlapping sets of taxa. The agreement problem asks whether there exists a tree {mathcal {T}}, called an agreement tree, whose taxon set is the union of the taxon sets of the input trees such that the restriction of {mathcal {T}} to the taxon set of {mathcal {T}}_i is isomorphic to {mathcal {T}}_i, for each i in {1, 2, ldots , k}. The agreement problems is a special case of the supertree problem, the problem of synthesizing a collection of phylogenetic trees with partially overlapping taxon sets into a single supertree that represents the information in the input trees. An obstacle to building large phylogenetic supertrees is the limited amount of taxonomic overlap among the phylogenetic studies from which the input trees are obtained. Incorporating taxonomies into supertree analyses can alleviate this issue. ResultsWe give a {mathcal {O}}(n k (sum _{i in [k]} d_i + log ^2(nk))) algorithm for the agreement problem, where n is the total number of distinct taxa in {mathcal {P}}, k is the number of trees in {mathcal {P}}, and d_i is the maximum number of children of a node in {mathcal {T}}_i.ConclusionOur algorithm can aid in integrating taxonomies into supertree analyses. Our computational experience with the algorithm suggests that its performance in practice is much better than its worst-case bound indicates.

Enhanced effects of walnut green husk solution on the phytoextraction of soil Cd and Zn and corresponding microbial responses

Walnut green husk (WGH) is a common agricultural waste, but it may be conducive to phytoremediation of heavy metals owing to its abundant phenolic hydroxyl, carboxyl, and other functional groups. In this study, WGH solution was used as an enhancer in the phytoextraction process via Sedum plumbizincicola, a hyperaccumulator of Zn and Cd. Microbial responses in the soil and plants were seamlessly analyzed to determine the underlying mechanisms of heavy metal extraction in this process. The results showed that the addition of 0.05 g/mL WGH solution increased the accumulation of Cd (by 153%) and Zn (by 220%) in plants as well as the availabilities of Cd (by 29.46%) and Zn (by 9.71%) in the soil, which can be attributed to an increase in the relative abundance of plant growth-promoting rhizobacteria that benefit phytoextraction. Furthermore, co-occurrence network analysis indicated the keystone taxa in the microbial community. Particularly, one of the keystone taxa, Pseudomonas, was also identified as distinct taxon in soil, and it was dominant among the endophytic bacteria. These results indicated that Pseudomonas, a taxon responded to WGH solution, may play a key role in enhanced phytoextraction in both soil and plant root. These results help to a better understanding of the microbial mechanisms for heavy metal accumulation in hyperaccumulator.

The impact of habitat loss on pollination services for a threatened dune endemic plant.

Habitat loss is currently a major threat to biodiversity, affecting species interactions, such as plant-pollinator interactions. This is particularly important in self-incompatible plants relying on pollinators to reproduce and sustain their populations. Here, we evaluated how habitat loss affects the pollination system, plant individual-pollinator species interaction network, and plant reproductive fitness of the self-incompatible Jasione maritima var. sabularia, a threatened taxon from dune systems. This plant is a pollinator generalist, visited by 108 species from distinct taxonomic groups. Results suggest that increasing habitat loss led to a significant decline in pollinator richness, increased pollen limitation, and a decrease in reproductive fitness of J. maritima var. sabularia. Visitation rate per individual did not significantly change with available area, indicating that the quality of pollen differed across populations. The topology of the network between J. maritima var. sabularia individuals and its pollinator species did not change, which may be attributed to the stability in the core of pollinator species. This suggests that the lower fitness of plants with increasing habitat degradation may be explained not only by the lower richness of peripheral pollinators but also by the genetic structure of the plant populations, as there is a possible higher transference of less quality pollen by pollinators, ultimately compromising the persistence of plant populations. Our study highlights the need of future studies to integrate the fine details provided by individual-level networks, which will increase our understanding of the pattern of species interactions and its consequences for the fitness of threatened plant populations.

Comparative cytogenetics of three economically important Piper L. species from the Brazilian Amazon.

The species Piper hispidinervum, Piper aduncum, and Piper affinis hispidinervum have essential oils with high levels of safrole, dillapiole, and sarisan, respectively. Safrole is important for pharmaceutical and chemical industries, while dillapiole and sarisan are promising compounds to control insects and fungi. These species are very similar morphologically and their taxonomy is controversial. Divergent hypotheses consider P. aduncum and P. hispidinervum either as a single species or as distinct taxa, while P. affinis hispidinervum is inferred to be a natural hybrid or a chemotype of P. hispidinervum. Delimiting the taxonomic boundaries would be helpful for germplasm conservation and breeding programs. This study aimed to undertake a detailed analysis of P. aduncum, P. hispidinervum, and P. affinis hispidinervum karyotype and rDNA sites. Genomic in situ hybridization (GISH) was used to establish genomic homology among species and to test the natural hybridization hypothesis for origin of P. affinis hispidinervum. Karyotype traits were similar for all three species: 2n = 26 small chromosomes, predominantly metacentric. All three species exhibited CMA+ bands on the secondary constriction of chromosome pair 4. A size-heteromorphic 35S rDNA site was co-localized with the CMA+ band. A 5S rDNA site was located in the proximal region of chromosome pair 7. The patterns of genomic hybridization revealed that the repetitive DNA fraction of the species is highly similar in terms of proportion of genome, sequence type, and distribution. Our findings did not allow us to differentiate the three species and point to the importance of deeper genomic studies to elucidate the taxonomic controversy.

Morphology and molecular phylogeny of Bindiferia gen. nov. (Dinophyceae), a new marine, sand-dwelling dinoflagellate genus formerly classified within Amphidinium

ABSTRACT As historically described, the genus Amphidinum has long been recognized to be polyphyletic. Amphidinium sensu lato is the most diverse of all marine benthic dinoflagellate genera and after the redefinition of the sensu stricto genus about 100 species remained of unknown generic affiliation. A species similar to the Australian sensu lato species Amphidinium boggayum was discovered in France. Morphological and molecular phylogenetic data supported the description of a new species and the classification of the two closely related species within a new athecate, photosynthetic, benthic genus, named Bindiferia. Cells were dorsoventrally flattened with smaller asymmetric epicone. The cingulum was descending. The sulcus reached the antapex and had an extension onto the epicone. The apical structure complex formed an anticlockwise loop around the apex. A red stigma was located in the anterior part of the epicone, near the apex. Vegetative division occurred in a non-motile stage within a hyaline sheath. Non-motile cells had dorsal hypocone grooves. The two species differed from each other in epicone size, cingulum displacement, nucleus location and surface groove occurrence. The molecular phylogenetic results showed that Bindiferia is a distinct taxon that is only distantly related to Amphidinium sensu stricto. The nearest sister group to Bindiferia could not be reliably determined, but the apical structure complex suggested a close relationship to the genera of the Gymnodinium sensu stricto clade.

Caveats of the internal transcribed spacer region as a barcode to resolve species boundaries in Diaporthe

Species in Diaporthe are largely reported as important plant pathogens. Identification of species in this genus has been complemented by morphological and molecular features. However, one important factor delaying this process is the struggle to formulate robust species concepts to create adequate international phytosanitary measures. Regardless of the wide use of the internal transcribed spacer (ITS) rDNA region, established as the primary DNA barcode for fungi, the tendency for intraspecific variation has been reported, misleading interpretation of phylogenetic analyses. Therefore, the present study aimed to illustrate, using specific examples, how the ITS region may be problematic for species delimitation. We showed that the ITS region is highly variable, with strains of Diaporthe malorum and Diaporthe novem falling into more than one clade, which if analyzed on their own, would be likely recognized as distinct taxa. Divergent ITS paralogs were also proven to coexist within the genome of D. novem. We also suggest that ITS may have escaped from concerted evolution or has undergone a duplication event. Furthermore, this study reports for the first time the existence of a putative hybrid in the genus Diaporthe. Our findings offer new clues towards the intraspecific and intragenomic variation in the ITS region, raising questions about its value for barcoding, i.e., identifying species in the genus Diaporthe. Therefore, we recommend that the ITS region be analyzed cautiously and always compared for congruence prior to description of novel taxa.

Primate body mass and dietary correlates of tooth root surface area.

This study aims to examine primate postcanine tooth root surface area (TRSA) in the context of two ecological variables (diet and bite force). We also assess scaling relationships within distinct taxonomic groups and across the order as a whole. Mandibular postcanine TRSA was measured using a three-dimensional computed tomography (CT) method for catarrhine (N=27), platyrrhine (N=21), and strepsirrhine (N=24) taxa; this represents the first sample of strepsirrhines. Two different body size proxies were used: cranial geometric mean (GM) using nine linear measurements, and literature-derived body mass (BM). TRSA correlated strongly with body size, scaling with positive allometry or isometry across the order as a whole; however, scaling differed significantly between taxa for some teeth. Among Strepsirrhini, molar TRSA relative to GM differed significantly between folivores and pliant-object feeders. Additionally, P4 TRSA relative to BM differentiated folivores from both hard- and pliant-object feeders. Among Cercopithecoidea, P4 TRSA adjusted by GM differed between hard- and pliant-object feeders. Dietary signals in TRSA appear primarily driven by high frequency loading experienced by folivores. Stronger and more frequent dietary signals were observed within Strepsirrhini relative to Haplorhini. This may reflect the constraints of orthognathism within the latter, constraining the adaptability of their postcanine teeth. Finally, because of the strong correlation between TRSA and BM for each tooth locus (mean r2 =0.82), TRSA can be used to predict BM in fossil primates using provided equations.

A taxonomic assessment of Chlorospingus flavopectus phaeocephalus and Chlorospingus semifuscus (Passeriformes: Passerellidae), including the description of a new subspecies.

Chlorospingus flavopectus, a widely distributed member of the New World sparrows and finches (Passerellidae), is among the most variable and complex Neotropical bird species. With up to 28 subspecies that inhabit montane forest from Mexico south to Argentina, it presents a recurring leapfrog pattern, with many genetically differentiated lineages, but even more morphologically distinguishable taxa. Chlorospingus flavopectus phaeocephalus is distributed along the eastern Andean slope (from southern Colombia to northern Peru) and in localized patches along the central and southwestern slopes of the Ecuadorian Andes, in the forest remnants of Chimborazo, El Oro, and adjacent Loja provinces. This allopatric distribution was based on plumage similarities, but no genetic or vocal analysis has tested if these populations share a common ancestry. Here, we compared data for populations of C. f. phaeocephalus, C. semifuscus semifuscus from northwest Ecuador, and C. s. livingstoni from west Colombia, and analyzed them in the context of other Chlorospingus. Our phylogenetic analyses revealed that the putative west Ecuadorian populations of C. f. phaeocephalus are nested within C. semifuscus. They also present diagnostic characters when compared to other populations of C. semifuscus and C. f. phaeocephalus. Based on these results, we recognize the population from El Oro and adjacent Loja Provinces as a new subspecies of C. semifuscus, and the population from Chimborazo Province as a morphological variation of C. s. semifuscus.

Integrative Study of Genotypic and Phenotypic Diversity in the Eurasian Orchid Genus Neotinea.

Knowledge of population variation across species’ ranges is a prerequisite for correctly assessing the overall variability of any group of organisms and provides an invaluable basis for unraveling evolutionary history, optimizing taxonomy and devising effective conservation strategies. Here, we examine the genus Neotinea, which represents a relatively recently delimited monophyletic genus of orchids, for which a detailed study of its overall variability was lacking. We applied a suite of biosystematic methods, consisting of flow cytometry, multivariate and geometric morphometrics, and analysis of genomic SNP data, to identify phylogenetic lineages within the genus, to delineate phenotypic variation relevant to these lineages, and to identify potential cryptic taxa within lineages. We found clear differentiation into four major lineages corresponding to the groups usually recognized within the genus: Neotinea maculata as a distinct and separate taxon, the Neotinea lactea group comprising two Mediterranean taxa N. lactea and Neotinea conica, the Neotinea ustulata group comprising two phenologically distinct varieties, and the rather complex Neotinea tridentata group comprising two major lineages and various minor lineages of unclear taxonomic value. N. conica constitutes both a monophyletic group within N. lactea and a distinct phenotype within the genus and merits its proposed subspecies-level recognition. By contrast, the spring and summer flowering forms of N. ustulata (var. ustulata and var. aestivalis) were confirmed to be distinct only morphologically, not phylogenetically. The most complex pattern emerged in the N. tridentata group, which splits into two main clades, one containing lineages from the Balkans and eastern Mediterranean and the other consisting of plants from Central Europe and the central Mediterranean. These individual lineages differ in genome size and show moderate degrees of morphological divergence. The tetraploid Neotinea commutata is closely related to the N. tridentata group, but our evidence points to an auto- rather than an allopolyploid origin. Our broad methodological approach proved effective in recognizing cryptic lineages among the orchids, and we propose the joint analysis of flow cytometric data on genome size and endopolyploidy as a useful and beneficial marker for delineating orchid species with partial endoreplication.