Two distinct, geographically overlapping lineages of the corallimorpharian Ricordea florida (Cnidaria: Hexacorallia: Ricordeidae)

We examined the genetic variation of the corallimorpharian Ricordea florida; it is distributed throughout the Caribbean region and is heavily harvested for the marine aquarium trade. Eighty-four distinct individuals of R. florida were sequenced from four geographically distant Caribbean locations (Cura?ao, Florida, Guadeloupe, and Puerto Rico). Analysis of the ribosomal nuclear region (ITS1, 5.8S, ITS2) uncovered two geographically partially overlapping genetic lineages in R. florida, probably representing two cryptic species. Lineage 1 was found in Florida and Puerto Rico, and Lineage 2 was found in Florida, Puerto Rico, Guadeloupe, and Cura?ao. Because of the multi-allelic nature of the ITS region, four individuals from Lineage 1 and six from Lineage 2 were cloned to evaluate the levels of hidden intra-individual variability. Pairwise genetic comparisons indicated that the levels of intra-individual and intra-lineage variability (<1%) were approximately an order of magnitude lower than the divergence (~9%) observed between the two lineages. The fishery regulations of the aquarium trade regard R. florida as one species. More refined regulations should take into account the presence of two genetic lineages, and they should be managed separately in order to preserve the long-term evolutionary potential of this corallimorpharian. The discovery of two distinct lineages in R. florida illustrates the importance of evaluating genetic variability in harvested species prior to the implementation of management policies.     

INTRAPOPULATIONAL VARIATION IN LEAFLET MORPHOLOGY OF ZAMIA PUMILA L. IN RELATION TO MICROENVIRONMENT AND SEX

The cycad Zamia pumila L. was examined for differences in leaflet morphology between sun and shade plants and between males and females within a single population in northern Puerto Rico. Sun leaflets are significantly smaller than shade leaflets in length, width, and surface area; and sun leaflets have a higher length: width ratio than shade leaflets. Also, average densitythickness is greater for sun leaflets than for shade leaflets. Males and females are similar in leaflet size; but the females had a larger number of leaflets per leaf than males.     

Origin of the green iguana (Iguana iguana) invasion in the greater Caribbean Region and Fiji

Invasive populations of green iguanas (Iguanidae: Iguana iguana) are widely established beyond their native Central, South American, and Lesser Antillean range in various islands of the Pacific, Florida USA, and in the Greater Caribbean Region. Although widespread, information about these invasions is scarce. Here we determine the origin of invasive populations of green iguanas in Puerto Rico, Fiji, The Caymans, Florida USA, The Dominican Republic, the US Virgin Islands (USVI) of St. Thomas and St. Croix, and a U.S.A pet store. We sampled 120 individuals from these locations and sequenced one mitochondrial (ND4) and two nuclear (PAC and NT3) loci. We also include a preliminary characterization of population structure throughout Puerto Rico using six microsatellite loci to genotype individuals across 10 sampling sites. Comparing the genealogical relationships of all our samples to published sequencing data from the native range, we found that sampled populations were largely a product of populations from Colombia and El Salvador; two countries with multiple, industrial-size pet iguana farming operations. Notably, we found that haplotypes detected exclusively in the USVI and Puerto Rico’s outlying island of Vieques are closely linked to green iguanas native to Saba and Montserrat (Lesser Antilles); a clade not reported in the pet trade. Our population genetic analyses did not reveal isolation among sampling sites in Puerto Rico, rather the evidence supported admixture across the island. This study highlights the roles of the pet trade and lack of regulation in the spread of green iguanas beyond their native range.

     

A review of the net-winged midges (Diptera: Blephariceridae) of the West Indies,with description of a new species from Puerto Rico

ABSTRACT

The Caribbean blepharicerid fauna comprises three described species: Paltostoma palominoi Hogue and Garces, 1990, found in eastern Cuba; P. agyrocincta Curran, 1927, widespread in Puerto Rico; and P. schineri Williston, 1896, which occupies most major islands of the Lesser Antilles. In this paper, we describe P. portoricensis Hogue and Courtney, sp. n., a second species from Puerto Rico. We also provide keys to adults, pupae, and larvae of known Caribbean species, re-describe P. argyrocincta and P. schineri, and present a brief discussion of the bionomics and phylogenetic affinities of the net-winged midge fauna.     

The Systematics and Reproductive Biology of the Galeus arae Subspecific Complex (Chondrichthyes: Scyliorhinidae)

Galeus arae is currently classified as a complex of three subspecies (Galeus arae arae, Galeus arae antillensis, and Galeus arae cadenati). Morphometric and meristic analyses, size at maturity, and variation in color patterns, support the recognition of these taxa as distinct species. All species have well-developed nidamental glands and are oviparous. Galeus arae comprises two geographically disjunct populations that are not distinguishable by the characters we examined. A northern population occurs along the east and Gulf coasts of North America from South Carolina to the Mississippi delta, and the northern coast of Cuba to the north-eastern tip of the Yucatan. A southern population occurs along the Caribbean coasts of Honduras, Nicaragua, and Costa Rica, and some neighboring islands. Galeus antillensis occurs on the northern coasts of Hispaniola and Puerto Rico, and off many of the Leeward Islands. Galeus cadenati occurs off the Caribbean coasts of Panama and Colombia. Distributional data suggest that the three species are distributed allopatrically.     

Further observations of blunt-hooked pentastomids belonging to the genus Raillietiella Sambon, 1910 infecting Hemidactylus brookii (Sauria: Gekkonidae) in Africa and the Caribbean: comparison with closely related Raillietiella spp. from an African skink (Mabuya perrotetii)

Hemidactylus brookii, a gecko possessing great dispersal ability, has apparently spread from West Africa to the Caribbean and South America by natural rafting. The lungs of populations from West Africa and the Greater Antilles (hispaniola and Puerto Rico) are heavily infected by raillietiellied pentastomids. Nigerian populations harbour two distinct species, frequently as concurrent infections, the males of which can be separated principally by marked differences in the morphology of the copulatory spicule. However one of these species cannot be reliably distinguished from Raillietiella teagueselfi, a common parasite of another highly successful coloniser, the Mediterranean gecko H. turcicus in Texas. The other more closely resembles R. frenatus, first described from the lungs of yet another itinerant gecko (H. frenatus) from south east Asia. Raillietiella spp. from both the Hispaniolan and Puerto Rican H. brookii haitianus populations, possess bluntly-rounded spicules of the R. frenatus type, although comparative posterior hook dimensions indicate that they are not closely related to it, or indeed to each other. They clearly comprise recognisably different geographical populations, but it remains unclear as to whether these are endemic or introduced. Both of the Caribbean species are left unnamed because we do not know what proportion of the observed intra- and interspecific variation is genetically based. Analysis of another raillietiellid population from an African skink (Mabuya perrotetii) is also included because it harbours two more species which are not easily distinguished from R. frenatus and R. teagueselfi. More refined diagnostic techniques, ultimately supplemented by life-cycle studies, are required to resolve these taxonomic problems.     

POPULATIONS OF MONARCH BUTTERFLIES WITH DIFFERENT MIGRATORY BEHAVIORS SHOW DIVERGENCE IN WING MORPHOLOGY

The demands of long‐distance flight represent an important evolutionary force operating on the traits of migratory species. Monarchs are widespread butterflies known for their annual migrations in North America. We examined divergence in wing morphology among migratory monarchs from eastern and western N. America, and nonmigratory monarchs in S. Florida, Puerto Rico, Costa Rica, and Hawaii. For the three N. American populations, we also examined monarchs reared in four common environment experiments. We used image analysis to measure multiple traits including forewing area and aspect ratio; for laboratory‐reared monarchs we also quantified body area and wing loading. Results showed wild monarchs from all nonmigratory populations were smaller than those from migratory populations. Wild and captive‐reared eastern monarchs had the largest and most elongated forewings, whereas monarchs from Puerto Rico and Costa Rica had the smallest and roundest forewings. Eastern monarchs also had the largest bodies and high measures of wing loading, whereas western and S. Florida monarchs had less elongated forewings and smaller bodies. Among captive‐reared butterflies, family‐level effects provided evidence that genetic factors contributed to variation in wing traits. Collectively, these results support evolutionary responses to long‐distance flight in monarchs, with implications for the conservation of phenotypically distinct wild populations.     

Morphological variation inBemisia endosymbionts

Summary The ultrastructure of the endosymbionts of several populations of whitefly (Homoptera: Aleyrodidae) was examined using transmission electron microscopy. Consistent differences in morphology and relative number of endosymbionts were observed between species and biotypes of whitefly within the Bemisia taxon.Bemisia argentifolii (=B. tabaci B biotype) individuals from Hawaii, Florida, and Arizona contained two morphological types of microorganisms housed within the mycetocyte cells of immature whiteflies. In contrast, individuals from populations ofB. tabaci A biotype from Arizona and Mexico, andB. tabaci Jatropha biotype from Puerto Rico, consistently contained three distinct morphological types of microorganisms within their mycetocytes. Organisms fromB. tabaci A and Jatropha biotypes differed from each other in the relative frequency of each type of microorganism. These observations suggest that different whitefly biotypes may have variable combinations of micro-fauna, with some possibly unique to each group, and furthers the hypothesis that variation in whitefly endosymbionts may be associated with the development of biotypes.     

The influence of spatial scale on the genetic structure of a widespread tropical wetland tree, Pterocarpus officinalis (Fabaceae)

Identifying factors that cause genetic differentiation in plant populations and the spatial scale at which genetic structuring can be detected will help to understand plant population dynamics and identify conservation units. In this study, we determined the genetic structure and diversity of Pterocarpus officinalis, a widespread tropical wetland tree, at three spatial scales: (1) drainage basin “watershed” (<10 km), (2) within Puerto Rico (<100 km), and (3) Caribbean-wide (>1000 km) using AFLP. At all three spatial scales, most of the genetic variation occurred within populations, but as the spatial scale increased from the watershed to the Caribbean region, there was an increase in the among population variation (Φ_ST=0.19 to Φ_ST=0.53). At the watershed scale, there was no significant differentiation (P=0.77) among populations in the different watersheds, although there was some evidence that montane and coastal populations differed (P<0.01). At the island scale, there was significant differentiation (P<0.001) among four populations in Puerto Rico. At the regional scale (>1000 km), we found significant differentiation (P<0.001) between island and continental populations in the Caribbean region, which we attributed to factors associated with the colonization history of P. officinalis in the Neotropics. Given that genetic structure can occur from local to regional spatial scales, it is critical that conservation recommendations be based on genetic information collected at the appropriate spatial scale.     

Genome sizes for 71 species of Zamia (Cycadales: Zamiaceae) correspond with three different biogeographic regions

Nuclear DNA contents (2C‐value) are reported for 71 out of 76 accepted species of Zamia (Zamiaceae) using flow cytometry with propidium iodide. Nuclear DNA content in Zamia ranges between 33.7 and 45.7 pg. Despite this small range, the largest genome contains roughly 10¹⁰ more base pairs than the smallest genome. The results for Zamia point to two centers of biogeographic distribution: Mexico and Colombia. Nicaragua seems to be the biogeographic boundary for these two centres for Zamia. To the north, genome sizes of 33.7–38.0 pg (average 35.6 pg) are found and to the south (Costa Rica, Panama and South America) 41.2–45.7 pg (average 42.9 pg). Plants from the Caribbean islands (including Florida) have intermediate genome sizes with 37.3–40.9 pg (average 38.7 pg). Costa Rica and Panama are in a transition zone and its species can be divided into three subsections: four species with ‘Caribbean’ values of 38.4–39.5 pg (average 39.0 pg), six species with ‘South American’ values with 42.7–43.6 pg, (average 42.9 pg, and six species with intermediate values ranging between 40.1–41.0 pg (average 40.4 pg). The latter values are nearly absent in other areas, suggesting that they could be the products of (introgressive) hybridization. This study represents the first, nearly complete overview of the genome sizes of the genus Zamia and their relationship with biogeography.     

HISTORICAL BIOGEOGRAPHY OF THE BANANAQUIT (COEREBA FLAVEOLA) IN THE CARIBBEAN REGION: A MITOCHONDRIAL DNA ASSESSMENT

We analyzed mitochondrial DNA (mtDNA) restriction-site variation in bananaquit (Coereba flaveola; Aves, Coerebinae) populations sampled on 12 Caribbean islands and at 5 continental localities in Central America and northern South America. Multiple fixed restriction-site differences genetically defined several regional bananaquit populations. An mtDNA clade representing all Jamaican bananaquits was the most divergent; the estimated average sequence divergence (d_xy) between Jamaican and all other mtDNA haplotypes surveyed was 0.027. Three groups of populations, representing Central America, northern South America, and the eastern Antilles (Puerto Rico to Grenada) were nearly equally differentiated among themselves (average d_xy = 0.014), and may represent a single, recent range expansion. Within the eastern Antilles, three geographically restricted haplotype groups were identified: Puerto Rico, north-central Lesser Antilles (U.S. Virgin Islands to St. Lucia), and Grenada–St. Vincent. The evolutionary relationships of these groups were not clear. Genetic homogeneity of the island populations from the U.S. Virgin Islands to St. Lucia suggested a recent spread of a specific north-central Lesser Antillean haplotype through most of those islands. Haplotype variation across this region indicated that this spread may have occurred in two waves, first through the southernmost islands of St. Lucia, Martinique, and Dominica, and more recently from Guadeloupe to the north. The geographic distribution of mtDNA haplotypes, and of bananaquit populations, suggests periods of invasiveness followed by relative geographic quiescence. Although most genetic studies of bird populations have revealed homogeneity over large geographic areas, our findings provide a remarkable counterexample of strong geographic structuring of mtDNA variation over relatively small distances. Furthermore, although the mtDNA data were consistent with several subspecific distinctions, it was clear that named subspecies do not define equally differentiated evolutionary entities.     

Circumtropical distribution of the epiphytic dinoflagellate Coolia malayensis (Dinophyceae): Morphology and molecular phylogeny from Puerto Rico and Brazil

The dinoflagellate genus Coolia, which contains potentially toxic species, is an important component of epiphytic assemblages in marine ecosystems. The morphology of C. malayensis has been illustrated from strains isolated in Asia and Oceania. In this study, strains of C. malayensis isolated from the Caribbean Sea in Puerto Rico, and for the first time from the South Atlantic Ocean in Brazil, were investigated by light, epifluorescence and scanning electron microscopies. No significant morphological differences between these new strains and other geographically distant strains of C. malayensis were observed. In the LSU rDNA phylogeny, the C. malayensis sequences from Brazil and Puerto Rico branched within the clade of strains from Oceania and Asia. The recently described species C. santacroce branched as a sister group of C. monotis, and C. palmyrensis was basal to the combined group of C. monotis/C. malayensis/C. santacroce. A tentative undescribed species from Florida and New Zealand branched as a sister group of C. malayensis. Our results confirm that C. malayensis showed a cosmopolitan distribution in tropical to subtropical waters, while the type species C. monotis remains endemic for the Mediterranean Sea and the temperate North Atlantic.     

Regeneration filters in post-agricultural fields of Puerto Rico and Ecuador

To better understand how rainforest regenerates after agriculture I sampled the seed rain and seed bank, and set out seeds and seedlings, on microsites defined by distance to the forest in fields both in Puerto Rico and Ecuador. I found that (1) total seeds, species richness and life-form richness were twice as great in the Ecuador seed rain compared to Puerto Rico but Puerto Rico seeds were more evenly distributed among species and (2) total seedlings from the seed bank were similar between Puerto Rico and Ecuador, (3) the majority of seeds were lost to predation among all species and study sites, (4) seed disease was absent in P. aduncum and Miconia prasina, and no seeds germinated for Gonzalagunia spicata and P. riparia, (5) in Ecuador pathogenic disease claimed more seeds than germinated for all species, and Solanum ovalifolium was the only species that had seeds germinate but did not lose seeds to disease, (6) also in Ecuador, insect predation was significantly lower in the forest border for P. aduncum, and seed disease was significantly greater at the 10 m micro site for S. ovalifolium, (7) distance has a significant effect on seedling height and basal diameter, (8) losses of leaf area due to herbivory and pathogens were always low and (9) biomass and leaf specific mass were significantly reduced in the border and forest microsites. I conclude that Ecuador fields had more seeds, species, and life-forms than Puerto Rico fields, predation was the most severe post-dispersal seed filter in all fields, seeds that survived predation on Puerto Rico were lost either to disease or germination but to both mechanisms in Ecuador, all three seed mechanisms in Ecuador fields showed distance effects of seedling growth but not survivorship.     

Colonization of islands in the Mona Passage by endemic dwarf geckoes (genus Sphaerodactylus) reconstructed with mitochondrial phylogeny

Little is known about the natural history of the Sphaerodactylus species endemic to the three islands located in the Mona Passage separating the Greater Antillean islands of Hispaniola and Puerto Rico. In this study, parts of two mitochondrial genes, 16S rRNA and 12S rRNA, were sequenced to determine the relationships between the sphaerodactylids that live in the Mona Passage and other Caribbean species from the same genus. While the main goal was to identify the biogeographical origin of these species, we also identified a genetically distinct type of dwarf gecko that warrants future evaluation as a possible new species. According to the reconstructed phylogenies, we propose a stepwise model of colonization wherein S. nicholsi from southwestern Puerto Rico or a very close ancestor gave rise through a founder event to Sphaerodactylus monensis on Mona Island. In a similar fashion, S. monensis or a very close ancestor on Mona Island gave rise to S. levinsi on Desecheo Island. This study also suggests that the most recent common ancestor between the species from the islands in the Mona Passage and Puerto Rico existed approximately 3 MYA.     

Demographic history and genetic diversity in West Indian Coereba flaveola populations

The bananaquit (Coereba flaveola) has been well studied throughout the Caribbean region from a phylogenetic perspective. However, data concerning the population genetics and long-term demography of this bird species are lacking. In this study, we focused on three populations within the Lesser Antilles and one on Puerto Rico and assessed genetic and demographic processes, using five nuclear and two mitochondrial markers. We found that genetic diversity of bananaquits on Puerto Rico exceeds that on the smaller islands (Dominica, Guadeloupe and Grenada); this might reflect either successive founder events from Puerto Rico to Grenada, or more rapid drift in smaller populations subsequent to colonization. Population growth rate estimates showed no evidence of rapid expansion and migration was indicated only between populations from the closest islands of Dominica and Guadeloupe. Overall, our results suggest that a "demographic fission" model, considering only mutation and drift, but without migration, can be applied to these bananaquit populations in the West Indies.     

Body size variation and caste ratios in geographically distinct populations of the invasive big‐headed ant,Pheidole megacephala (Hymenoptera: Formicidae)

Body size is an important life history trait that can evolve rapidly as a result of how species interact with each other and their environment. Invasive species often encounter vastly different ecological conditions throughout their introduced range that can influence relative investment in growth, reproduction and defence among populations. In this study, we quantified variation in worker size, morphology and proportion of majors among five populations of a worldwide invasive species, the big‐headed ant, Pheidole megacephala (Fabricius). The sampled populations differed in ant community composition, allowing us to examine if P. megacephala invests differently in the size and number of majors based on the local ant fauna. We also used genetic data to determine if these populations of P. megacephala represented cryptic species or if morphological differences could be attributed to change following introduction. We found significant variation in worker mass among the populations. Both major and minor workers were largest in Australia, where the ant fauna was most diverse, and minor workers were smallest in Hawaii and Mauritius, where P. megacephala interacted with few to no other ants. We also found differences in major and minor worker morphology among populations. Majors from Mauritius had significantly larger heads (width and length) relative to whole body size than those from Hawaii and Florida. Minors had longer heads and hind tibias in South Africa compared with populations from Australia, Hawaii and Florida. The proportion of majors did not differ among populations, suggesting that these populations may not be subject to trade‐offs in investment in major size versus number. Our molecular data place all samples within the same clade, supporting that these morphologically different populations represent the same species. These results suggest that the variation in shape and morphology of major and minor workers may therefore be the result of rapid adaptation or plastic responses to local conditions. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 423–438.     

小叶锦鸡儿天然居群叶形态性状变异研究

为揭示小叶锦鸡儿(Caragana microphylla)天然居群叶形态性状的变异规律及其生态适应性特征,该研究以10个小叶锦鸡儿天然居群为对象,通过多重比较、巢式方差分析、相关性分析、聚类分析和主成分分析等方法,对7个叶形态性状进行分析。结果表明：(1)小叶锦鸡儿叶形态性状在居群内和居群间均存在极显著差异(P < 0.01),平均变异系数为10.13%,不同性状的变异幅度为6.23%~12.78%;平均叶形态性状的表型分化系数为43.62%,居群内变异(30.09%)大于居群间变异(24.91%),说明居群内是其叶形态性状变异的主要来源。(2)相关性分析表明,环境因子对小叶锦鸡儿的叶形态性状变异有很大的影响,在地理空间上主要呈现出沿海拔梯度的变异模式;主成分分析的结果显示,小叶宽、叶柄宽和叶柄长对小叶锦鸡儿叶形态变异起主导作用;利用欧式距离对小叶锦鸡儿居群进行UPGMA聚类分析结果显示,基于叶形态性状和环境因子可分别将小叶锦鸡儿10个居群分为3类和2类,Mantel检验结果表明,小叶锦鸡儿的叶形态性状变异不存在地理连续性。研究结果为小叶锦鸡儿的适应性进化和开发利用提供了理论依据。     

Chemical control of the red palm mite, Raoiella indica (Acari: Tenuipalpidae) in banana and coconut

The red palm mite (RPM), Raoiella indica Hirst, is a predominant pest of coconuts, date palms and other palm species, as well as a major pest of bananas (Musa spp.) in different parts of the world. Recently, RPM dispersed throughout the Caribbean islands and has reached both the North and South American continents. The RPM introductions have caused severe damage to palm species, and bananas and plantains in the Caribbean region. The work presented herein is the result of several acaricide trials conducted in Puerto Rico and Florida on palms and bananas in order to provide chemical control alternatives to minimize the impact of this pest. Spiromesifen, dicofol and acequinocyl were effective in reducing the population of R. indica in coconut in Puerto Rico. Spray treatments with etoxanole, abamectin, pyridaben, milbemectin and sulfur showed mite control in Florida. In addition, the acaricides acequinocyl and spiromesifen were able to reduce the population of R. indica in banana trials.     

Saurian malaria in the Caribbean: Plasmodium azurophilum sp. nov., a malarial parasite with schizogony and gametogony in both red and white blood cells

A survey of 466 Caribbean lizards found Plasmodium parasites present in Anolis species only of five islands. Parasites presently considered to be P. floridense occurred on Grand Cayman, North Bimini, Jamaica, Hispaniola (Haiti), and Puerto Rico. A second species, P. azurophilum, is described as new from Anolis cybotes of Haiti, A. krugi of Puerto Rico, and A. lineatopus and A. grahami of Jamaica. It lacks visible pigment in erythrocytic host cells but can produce it occasionally. Both asexual and sexual forms occur in a variety of white blood cells, notably in azurophil granulocytes and polymorphonuclear leucocytes. Experimental infections indicate that the leucocytic phase occurs after the acute erythrocytic infection declines, thus suggesting that the schizogonic and gametogonic cycles in white cells may represent an adaptive defense against immune mechanisms of the host. Mean numbers of nuclei in schizonts and mean gametocyte size are influenced by host species and type of host cell.     

Analysis of host preference and geographical distribution of Anastrepha suspensa (Diptera: Tephritidae) using phylogenetic analyses of mitochondrial cytochrome oxidase I DNA sequence data

Anastrepha suspensa (Loew) is an economically important pest, restricted to the Greater Antilles and southern Florida. It infests a wide variety of hosts and is of quarantine importance in citrus, a multi-million dollar industry in Florida. The observed recent increase in citrus infested with A. suspensa in Florida has raised questions regarding host-specificity of certain populations and genetic diversity of the pest throughout its geographical distribution. Cytochrome oxidase I (COI) DNA sequence data was used to characterize the genetic diversity of A. suspensa from Florida and Caribbean populations reared from different host plants. Maximum likelihood and Bayesian phylogenetic methods were used to analyse COI data. Sequence variation among mitochondrial COI genes from 107 A. suspensa samples collected throughout Florida and the Caribbean ranged between 0 and 10% and placed all A. suspensa as a monophyletic group that united all A. suspensa in a clade sister to a Central American group of the A. fraterculus paraphyletic species complex. The most likely tree of the COI locus indicated that COI sequence variation was too low to provide resolution at the subspecies level, therefore monophyletic groups based on host-plant use, geography (Florida, Jamaica, Cayman Islands, Puerto Rico or Dominican Republic) or population sampled are not supported. This result indicates that either no population segregation has occurred based on these biological or geographical distinctions and that this is a generalist, polyphagous invasive genotype. Alternatively, if populations are distinct, the segregation event was more recent than can be distinguished based on COI sequence variation.