Complex origin of Trinitario-type Theobroma cacao (Malvaceae) from Trinidad and Tobago revealed using plastid genomics

Trinidad and Tobago has a long history of producing high-quality cacao (Theobroma cacao L.). Cacao genotypes in Trinidad and Tobago are of a highly distinctive kind, the so-called “Trinitario” cultivar group, widely considered to be of elite quality. The origin of Trinitario cacao is unclear, although it is generally considered to be of hybrid origin. We used massive parallel sequencing to identify polymorphic plastidic single nucleotide polymorphisms (cpSNPs) and polymorphic plastidic simple sequence repeats (cpSSRs) in order to determine the origin of the Trinitario cultivar group by comparing patterns of polymorphism to a reference set of ten completely sequenced chloroplast genomes (nine T. cacao and one outgroup, T. grandiflorum (Willd. ex Spreng.) Schum). Only three cpSNP haplotypes were present in the Trinitario cultivars sampled, each highly distinctive and corresponding to reference genotypes for the Criollo (CRI), Upper Amazon Forastero (UAF) and Lower Amazon Forastero (LAF) varietal groups. These three cpSNP haplotypes likely represent the founding lineages of cacao to Trinidad and Tobago. The cpSSRs were more variable with eight haplotypes, but these clustered into three groups corresponding to the three cpSNP haplotypes. The most common haplotype found in farms of Trinidad and Tobago was LAF, followed by UAF and then CRI. We conclude that the Trinitario cultivar group is of complex hybrid origin and has derived from at least three original introduction events.     

Matriarchal genetic population structure of North American beluga whales Delphinapterus leucas (Cetacea: Monodontidae)

The North American beluga whale Delphinapterus leucas population has been divided into a number of putative geographical stocks based upon migration routes and areas of summer concentration. Nucleotide sequences of the mitochondrial DNA (mtDNA) control region were used to assess whether these geographical stocks are genetically distinct. Beluga whale samples from 25 sites were collected primarily from aboriginal subsistence hunts across North America from 1984 to 1994. Thirty-nine mtDNA haplotypes were identified in 628 beluga samples. No differences were found in the distribution of haplotypes between male and female beluga whales at any sampling site. These haplotypes segregated into two distinct assemblages in both a haplotype network and a neighbour-joining tree. The haplotype assemblages had a geographically disjunct distribution that suggests postglacial recolonization of the North American Arctic from two different refugia.
An analysis of molecular variance based on haplotype relationships and frequency indicated genetic heterogeneity among beluga whale summering groups ( P ≤ 0.001). Sequence divergence estimates between sampling sites also indicated geographical differentiation, particularly between samples taken at east Hudson Bay or St Lawrence River and the western or central Arctic. The results of this study show a high degree of philopatry to specific summering areas by this highly mobile animal.     

MITOCHONDRIAL DNA SEQUENCE VARIATION AMONG NATURAL POPULATIONS OF THE TRINIDAD GUPPY,POECILIA RETICULATA

Guppies were sampled from eight populations representing four river drainage basins in northern Trinidad, and from one population on the nearby island of Tobago. For each individual, a 465 base pair (bp) segment of the control region of the mitochondrial genome was sequenced. The resulting DNA sequences were subjected to sequence divergence calculations and the populations were linked by maximum parsimony analysis to determine their phylogenetic relationships. Mitochondrial DNA (mtDNA) sequence variation was found both within and between river drainages, correlated with the geographic features of northern Trinidad. The variation observed exists primarily between drainages, particularly between the Oropuche drainage and all other Trinidad drainages examined. Estimates of time of divergence between guppy populations of different drainages, based on mtDNA sequence variation, ranged from 100,000 to 200,000 for the most recently separated populations and from 600,000 to 1.2 million years between the Oropuche populations and all others examined. Examination of fish from northeastern South America will be required to determine whether these populations differentiated in their present locations or were the result of separate invasions of Trinidad from different Venezuelan sources. However, genetic isolation of these populations appears to predate the current physical separation of the island of Trinidad from the Venezuelan mainland.     

Mitochondrial DNA reveals different phylogeographic structures in the water shrews Neomys anomalus and N. fodiens (Insectivora: Soricidae) in Europe

We studied the intraspecific genetic variation of 17 Neomys anomalus (Cabrera, 1907) (eight localities) and 24 Neomys fodiens (Pennant, 1771) (nine localities) in Europe. As molecular marker, we used the sequences of the mtDNA gene for cytochrome b (1140 bp from 24 specimens; 272 bp from 15 specimens). The phylogenetic analysis of N. fodiens shows slight divergence among haplotypes, with only one supported clade including two geographically distant haplotypes from Calabria (southern Italy) and Pyrenees (France); these haplotypes diverge from the others by 2.9% and 2.1%, respectively. In N. anomalus , one haplotype from Spain is very different, diverging from all others by 8–10% of the sequence. This level of divergence is similar to that found between N. anomalus and another species, Neomys teres . There are two other supported clades in N. anomalus , an Italian subclade and an eastern subclade comprising haplotypes from Turkey and Slovenia. Moreover, we found a different genetic structure between the two species with greater divergence in N. anomalus than in N. fodiens. The phylogeographic patterns are discussed in a temporal framework and considering the different ecological characteristics of these two species.     

Population genetic structure of the seed pathogen Pyrenophora semeniperda on Bromus tectorum in western North America

We examined genetic variation in the ascomycete pathogen Pyrenophora semeniperda cultured from seeds of the invasive grass Bromus tectorum in the Intermountain West of North America. We sequenced the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA genome in 417 monoconidial cultures collected from 20 sites in Washington, Idaho, Utah and Colorado, USA. ITS sequence diversity was surprisingly high; 12 unique haplotypes were identified, averaging 1.3% pairwise sequence divergence. All sites had at least two haplotypes present, and three sites had seven or more. One haplotype composed 60% of the isolates and occurred at all 20 locations; the remaining haplotypes generally occurred at low frequencies within sites but at multiple sites throughout the region. Sites in Washington and Idaho were more diverse than those in Utah and Colorado, averaging two more haplotypes and 67% more pairwise differences among haplotypes at a site. Analysis of molecular variance (AMOVA) indicated that more than 80% of the genetic variation was found within sampling locations, while 7-11% of the variation can be attributed to differences between northern (Washington and Idaho) and southern (Utah and Colorado) populations. The wide distribution of even uncommon haplotypes among sampling sites and weak correlations between genetic and geographic distances among populations (< 0.2) suggested that these populations recently were established from a common source. We hypothesize that the strains of P. semeniperda infecting B. tectorum in western North America probably arrived with the invasive grass from its native Eurasian range.     

A comparative study of the prevalence of the metabolic syndrome and its components in type 2 diabetic patients in two Caribbean islands using the new International Diabetes Federation definition

BACKGROUND AND AIM: Tobago and Trinidad are two Caribbean islands with distinct genetic background and lifestyles; while Tobago is serene and a tourist centre, Trinidad is characterized by a hustling and bustling lifestyle. The study was aimed at determining and comparing the prevalence of the metabolic syndrome (MetS) and its critical components in type 2 diabetic patients using the new International Diabetes Federation (IDF) definition. METHODS: Four hundred and thirteen (166 Tobago, 247 Trinidad) type 2 diabetic patients visiting 10 lifestyle disease clinics were studied. Blood pressure, anthropometric parameters (height, weight, body mass index and waist circumference) and overnight fasting blood samples were taken. Plasma glucose and serum triglycerides, total cholesterol, LDL- and HDL-cholesterol, insulin, and adiponectin were determined. Insulin resistance (IR) was determined using the HOMA method. RESULTS: The patients in Tobago were significantly older than patients in Trinidad (p < 0.001) but the duration of diabetes (9.4 +/- 0.5 vs. 11.1 +/- 0.7 yr), medications, generalized (31.7 vs. 38.8%) and central (78.5 vs. 83.7%) obesity were similar (p > 0.05). In comparison with patients in Tobago, diabetic patients in Trinidad, irrespective of gender, had significantly higher prevalence of IDF critical components such as raised BP, raised triglycerides and reduced HDL-cholesterol (all, p < 0.001). Thus, while more patients in Trinidad were diagnosed with MetS based on three or four components, more patients in Tobago were diagnosed based on two components (p < 0.001). CONCLUSIONS: There were high prevalence rates of the components of the MetS in both the islands of Tobago and Trinidad. Quantitatively, the aggregation of the components is higher in patients in Trinidad, which constitute greater risk for adverse cardiovascular outcome. Controlling central obesity should be the target in preventing MetS in the two islands.     

Genetic differentiation in the Trinidad endemic Mannophryne trinitatis (Anura: Aromobatidae): Miocene vicariance,in situ diversification and lack of geographical structuring across the island

Trinidad offers a unique study system within the Caribbean to assess the processes and patterns of amphibian speciation. We used mitochondrial DNA sequences to investigate the phylogenetic relationships and patterns of intraspecific genetic variation of Mannophryne trinitatis from Trinidad. Molecular clock estimates point to a genetic split between M. trinitatis and its sister species, M. venezuelensis, dating to the Late Miocene (c. 7–8 Mya), suggesting vicariance as a means of speciation when Trinidad pulled apart from northern Venezuela. M. trinitatis phylogenetic population analyses from ten Northern Range and four Central Range localities recovered three well‐resolved clades: a larger clade formed by haplotypes from Northern Range localities and two additional clades, one formed by haplotypes from the Central Range and another including haplotypes from Northern Range localities and one haplotype from the Central Range. Overall, our results show that the genetic diversity in M. trinitatis is not geographically structured but it is distributed among the various Northern and Central Range localities. In congruence with the vicariance speciation hypothesis, we attribute M. trinitatis present distribution and lack of genetic structure to multiple admixture events caused by climate changes that severely affected the topology of Trinidad throughout the Pliocene/Pleistocene periods.     

Divergence and Remarkable Diversity of the Y Chromosome in Guppies

The guppy sex chromosomes show an extraordinary diversity in divergence across populations and closely related species. In order to understand the dynamics of the guppy Y chromosome, we used linked-read sequencing to assess Y chromosome evolution and diversity across upstream and downstream population pairs that vary in predator and food abundance in three replicate watersheds. Based on our population-specific genome assemblies, we first confirmed and extended earlier reports of two strata on the guppy sex chromosomes. Stratum I shows significant accumulation of male-specific sequence, consistent with Y divergence, and predates the colonization of Trinidad. In contrast, Stratum II shows divergence from the X, but no Y-specific sequence, and this divergence is greater in three replicate upstream populations compared with their downstream pair. Despite longstanding assumptions that sex chromosome recombination suppression is achieved through inversions, we find no evidence of inversions associated with either Stratum I or Stratum II. Instead, we observe a remarkable diversity in Y chromosome haplotypes within each population, even in the ancestral Stratum I. This diversity is likely due to gradual mechanisms of recombination suppression, which, unlike an inversion, allow for the maintenance of multiple haplotypes. In addition, we show that this Y diversity is dominated by low-frequency haplotypes segregating in the population, suggesting a link between haplotype diversity and female preference for rare Y-linked color variation. Our results reveal the complex interplay between recombination suppression and Y chromosome divergence at the earliest stages of sex chromosome divergence.     

Genetic diversity and historical population structure in the New Zealand mayfly Acanthophlebia cruentata

1. Nucleotide sequences of a 280 base pair region of the cytochrome b gene were used to assess genetic diversity and to infer population histories in the New Zealand mayfly Acanthophlebia cruentata. 2. A hierarchial examination of populations from 19 streams at different spatial scales in the central and northern North Island of New Zealand found 34 haplotypes. A common haplotype was found in all central region streams and unique haplotypes in northern streams. Several central streams had region specific haplotypes with genetically differentiated populations at the 70–100 km scale. 3. Haplotype diversity was high (0.53–0.8) at most sites, but low (0–0.22) in some central sites. amova analyses found significant genetic diversity among regions (69%) and among catchments (58%). Most population pairwise F_ST tests were significant, with non‐significant pairwise tests among sites in the central region and pairs of sites between neighbouring streams. 4. The levels of sequence divergence are interpreted as the result of Pleistocene divergence in multiple refugia, leading to the evolution of regionally unique haplotypes. The low diversity in some central region populations may result from recent colonisation following local extinctions, associated with volcanic events.     

Ecology,conservation status,and phylogenetic placement of endemic Pristimantis frogs (Anura: Craugastoridae) in Trinidad and Tobago and genetic affinities to northern Venezuela

Trinidad and Tobago are home to three endemic species in the anuran genus Pristimantis, of which two (Pristimantis charlottevillensis and Pristimantis turpinorum) occur in Tobago alone and the third (Pristimantis urichi) is present on both islands. Earlier, the IUCN assessed the conservation status of these species as: P. urichi, Endangered (EN); P. charlottevillensis, Least Concern (LC); P. turpinorum, Vulnerable (VU). However, these assessments were based on very little field-based evidence. Here, we present survey results which contributed to reassessments as LC, VU and Data Deficient for these three species, respectively. Despite the close proximity of Trinidad to northern Venezuela, the islands do not share any Pristimantis species with the mainland, which holds a rich endemicity of Pristimantis regionally. In this study, we used genetic sequencing from several island populations and compared them to northern Venezuelan endemics to assess genetic divergence for the first time. The time tree analyses found that only the northern Tobago species P. turpinorum is closely related to mainland Pristimantis, with a genetic split dating to the Late Miocene, suggesting a vicariant event of mainland and island species. Pristimantis urichi, although identical between the two islands, remains highly divergent from the mainland species. Similar results were found for P. charlottevillensis. In addition, there was a high level of divergence between P. urichi and P. charlottevillensis. These findings indicate different island colonization events by different lineages. Sequencing other Venezuelan species remains pivotal to unravel the complexity of the colonization episodes in the region, likely influenced by the changing topography and multiple connection and isolation episodes of the islands by eustatic sea-level changes.     

Macrophyte development in unimpacted lowland rivers in Poland

Three ecologically and morphologically distinct forms of Arctic charr (Salvelinus alpinus L.) have been identified in Loch Rannoch, Scotland, whose evolutionary status and origins are incompletely understood. A study was made of restriction fragment length polymorphism (RFLPs) detected variation in the D-loop, ND1 and cytochrome b regions of the mitochondrial genome, encompassing >3500 bp. Eight RFLP haplotypes were identified that clustered into three distinct clans based on restriction differences and into four clans based on sequence differences. Significant differences in RFLP frequencies were found among all morph groups. The pelagic morph was highly divergent from the two benthic forms, with the benthic forms having variants from only one genetic clan while the pelagic was dominated by a single variant from another clan. The relative divergence observed among benthic and pelagic forms is ~10 fold greater when nucleotide divergence among the haplotypes, as well as haplotype frequency differences, is taken into account. Sequence divergence between haplotypes in the two main clans is of a similar order to that between haplotypes in these clans and a charr from North America. In contrast, divergence among the two benthic morphs relates entirely to differences in haplotype frequencies. The study confirms the genetic distinctiveness of the pelagic and benthic forms as well as of the two benthic forms. It strongly supports previous evidence that the genetic divergence between the pelagic and benthic populations is allopatric in origin. Additionally, the results strongly suggest that the two benthic populations have undergone peripatric divergence through the sequential colonisation of the two basins by one lineage, followed by their spatial separation and reproductive isolation.     

DNA barcoding of Cuban freshwater fishes: evidence for cryptic species and taxonomic conflicts

Despite ongoing efforts to protect species and ecosystems in Cuba, habitat degradation, overuse and introduction of alien species have posed serious challenges to native freshwater fish species. In spite of the accumulated knowledge on the systematics of this freshwater ichthyofauna, recent results suggested that we are far from having a complete picture of the Cuban freshwater fish diversity. It is estimated that 40% of freshwater Cuban fish are endemic; however, this number may be even higher. Partial sequences (652 bp) of the mitochondrial gene COI (cytochrome c oxidase subunit I) were used to barcode 126 individuals, representing 27 taxonomically recognized species in 17 genera and 10 families. Analysis was based on Kimura 2-parameter genetic distances, and for four genera a character-based analysis (population aggregation analysis) was also used. The mean conspecific, congeneric and confamiliar genetic distances were 0.6%, 9.1% and 20.2% respectively. Molecular species identification was in concordance with current taxonomical classification in 96.4% of cases, and based on the neighbour-joining trees, in all but one instance, members of a given genera clustered within the same clade. Within the genus Gambusia, genetic divergence analysis suggests that there may be at least four cryptic species. In contrast, low genetic divergence and a lack of diagnostic sites suggest that Rivulus insulaepinorum may be conspecific with Rivulus cylindraceus. Distance and character-based analysis were completely concordant, suggesting that they complement species identification. Overall, the results evidenced the usefulness of the DNA barcodes for cataloguing Cuban freshwater fish species and for identifying those groups that deserve further taxonomic attention.     

Diversity of mud-dauber wasps (Hymenoptera: Crabronidae: Trypoxylon Latreille) in a secondary forest of Trinidad,West Indies

Over a two-year period we operated two intercept traps for flying insects near and within a well-developed secondary forest in the Arima Valley of Trinidad. These yielded 368 Trypoxylon, representing 30 species. The sample’s Shannon–Wiener diversity of H′ = 2.61 is higher than that from a comparable study of Trypoxylon on the smaller island of Tobago (15 species, H′ = 2.09) and the even smaller Little Tobago (six species, H′ = 1.41). Analysis of the samples by the Chao1 method suggests that the species numbers recorded on the two smaller islands are complete, while there is at least one additional species at the Trinidad locality. The four most abundant species in our samples show no evident bias in numbers of females between the wet and dry seasons, consistent with the hypothesis that at least these species nest throughout the year.     

Paralogous mitochondrial control region in the giant tiger shrimp, Penaeus monodon (F.) affects population genetics inference: A cautionary tale

The mitochondrial control region (mtCR) is a widely used genetic marker for phylogenetic, phylogeographic and population genetic inference. The analysis of mtCR in 115 Indonesian specimens of the giant tiger shrimp, Penaeus monodon, revealed 26 individuals yielding a second - apparently paralogous - sequence in addition to the putatively authentic mitochondrial haplotype. The paralogous haplotypes fell into two major haplogroups that are highly diverged with respect to the authentic mitochondrial haplotypes (average pairwise sequence divergence of 12.5% and 5.0%, respectively). A comparison with published mtCR sequences of P. monodon showed that the paralogous contaminant sequences were inadvertently included in a series of recent population genetic studies, leading to seriously compromised conclusions about genetic diversity and differentiation. The prevalence of the paralogous haplotypes throughout the sampled Indo-Pacific populations is highly skewed: From African and Indian individuals only paralogs have been sequenced, while they are completely absent from Australian individuals. This suggests that geographically unequally distributed allelic variants at binding sites of the primer pair ordinarily used to amplify mtCR in P. monodon suppressed the amplification of authentic mtCR in a wide range of samples.     

The structure and interrelationship of fungal populations in native and cultivated soils

Mitochondrial DNA haplotypes have been characterized for 120 isolates of the asexual fungus Fusarium oxysporum. Sixty of these isolates were obtained from soil in a native grassland in the San Joaquin Valley of California, including 20 isolates from each of six different sampling locations. The same sampling strategy was used to obtain 60 additional isolates from an agricultural field of the same soil type directly adjacent to the native soil. Twenty-three different mitochondrial DNA haplotypes were identified among the 120 isolates, including 11 haplotypes represented by two or more isolates and 12 that were unique. The five most common mitochondrial DNA haplotypes accounted for 93 (78%) of the 120 isolates. Isolates representing each of these five mitochondrial DNA haplotypes were found both in the cultivated and in the native soil. Seventy-two per cent of the isolates found in the cultivated soil were associated with the same mitochondrial DNA haplotype as one or more isolates in the native soil. The remaining isolates in the cultivated soil were associated with comparatively rare mitochondrial DNA haplotypes, most of which showed a close relationship to one of the haplotypes found in the native soil. Hierarchial gene diversity analysis indicated that a significant proportion of the mitochondrial DNA haplotype diversity was attributable to differences between sampling sites in the native soil but not in the cultivated soil. This may reflect significant spatial structuring of genetic diversity in populations of F. oxysporum in a native soil. The proportion of mtDNA haplotype diversity attributable to differences between populations in the native and cultivated soils was not significant. This suggests that our entire collection, encompassing strains from both native and cultivated soils, is representative of a single population of F. oxysporum.     

Phylogeography of West Indies Coral snakes (Micrurus): Island colonisation and banding patterns

In this study, we analyse New World coral snakes in a phylogenetic framework based upon an increased molecular data set, including novel sequences for the only two sympatric species known from an island (Trinidad, West Indies). Their presence in Trinidad and absence in Tobago offers a unique system to study the phylogeography of the region. We assess the tempo and mode of colonisation of Micrurus on the island, in addition to discussing the phylogenetic relationships for the genus Micrurus concerning two phenotypic traits, body and tail banding patterns. These relationships are analysed for the first time on statistical coalescent phylogeographic discrete ancestral reconstruction. We find a robust phylogenetic component in these characteristics, where strongly supported clades are recovered: prior to the onset of the Early Miocene, a triadal and tricolour tail clade composed of species from South America, and a second clade dating to the Middle‐Late‐ Miocene with monadal and bicolour tails widely distributed from North to South America. The divergence between clades dates to the Oligocene and suggests an ancient pre‐isthmus divergence supporting the arrival of the triadal clade into South America, before the connection between Central and South America was established. We find the two coral snakes present in the West Indies, M. diutius and M. circinalis, belong to the triadal and monadal clades, respectively. Guyana and Trinidad Micrurus diutius share the same haplotypes suggesting a Late Pleistocene–Holocene vicariance when sea level rises separated Trinidad from the mainland. A second lineage of diutius‐like snakes is present in Guyana and is confirmed as M. lemniscatus which is assigned as a voucher and restricts the type locality for M. lemniscatus.     

Phylogeographic analysis of the brown alga Cutleria multifida (Tilopteridales,Phaeophyceae) suggests a complicated introduction history

In depth genetic comparisons of populations of Cutleria multifida (Tilopteridales, Phaeophyceae) collected from Europe, the northwestern Pacific Ocean, Australia and New Zealand using the DNA sequences of four gene regions (the mitochondrial cox2 and cox3 genes, the intergeneric spacer region adjacent to cox3, and the open reading frame) suggested that the northwestern European and Japanese populations were considerably greater in terms of their genetic divergence than Mediterranean, Australian or New Zealand populations. The haplotypes of the populations in northwestern European (distribution range including the type locality, seven haplotypes) and Japanese populations (seven haplotypes) were unique except for one shared haplotype. There were weak but positive correlations between the geographical distance and the genetic divergence among northwestern European and Japanese populations. Moreover, both female and male gametophytes occurred in eight of the nine Japanese localities, suggesting Japanese populations showed normal sexual heteromorphic life history of the species. In light of these results, it appears that Japanese populations were native to the area despite earlier hypothesis. In contrast, Australian and New Zealand populations were composed of only one haplotype that is very close to those found in northwestern Europe and Japan, suggesting a recent introduction history from Europe (or from northeastern Asia via Europe) by ship transport to Australia and New Zealand. The Mediterranean populations included two haplotypes identical to those found in northwestern Europe and Japan, and it is suggestive of transoceanic introductions of some populations between Mediterranean and Japanese coasts.     

Chloroplast microsatellite primers for cacao (Theobroma cacao) and other Malvaceae

? Premise of the study: Chloroplast microsatellites were developed in Theobroma cacao to examine the genetic diversity of cacao cultivars in Trinidad and Tobago. ? Methods and Results: Nine polymorphic microsatellites were designed from the chloroplast genomes of two T. cacao accessions. These microsatellites were tested in 95 hybrid accessions from Trinidad and Tobago. An average of 2.9 alleles per locus was found. ? Conclusions: These chloroplast microsatellites, particularly the highly polymorphic pentameric repeat, were useful in assessing genetic variation in T. cacao. In addition, these markers should also prove to be useful for population genetic studies in other species of Malvaceae.     

新疆草兔的种群遗传结构和亚种分化

新疆草兔 (Lepus capensis) 的群体遗传结构至今无系统的研究报道,亚种水平的分类也长期存在争议.该文测定了形态分类上的新疆草兔3个亚种共87个个体的线粒体DNA (mtDNA)控制区(control region,D-Loop)592 bp的序列,经分析发现148个多态性位点,共定义了44个单倍型.新疆草兔的单倍型多样度(h,0.977 ± 0.005)和核苷酸多样度(π,0.064 ± 0.031)都较高,显示了较高的遗传多样性.分子变异分析(AMOVA)结果显示,4个地理群体间的显著分化可能是由地理隔离造成的.群体遗传结构分析显示,新疆草兔包含4个进化枝,并且每个进化枝都对应特定的分布区域,显示了明显的系统地理结构.该研究的结果支持形态分类上草兔西域亚种(L.c.lehmanni)的分类地位; 但中亚亚种(L.c.centrasiaticus)被分为两个独立的进化枝,提示可能存在两个亚种; 帕米尔亚种(L.c.pamirensis)与其他亚种间的遗传距离在13%以上,提示其可能已达到种的分化水平.