The phylogeography of the Placozoa suggests a taxon‐rich phylum in tropical and subtropical waters

Placozoa has been a key phylum for understanding early metazoan evolution. Yet this phylum is officially monotypic and with respect to its general biology and ecology has remained widely unknown. Worldwide sampling and sequencing of the mitochondrial large ribosomal subunit (16S) reveals a cosmopolitan distribution in tropical and subtropical waters of genetically different clades. We sampled a total of 39 tropical and subtropical locations worldwide and found 23 positive sites for placozoans. The number of genetically characterized sites was thereby increased from 15 to 37. The new sampling identified the first genotypes from two new oceanographic regions, the Eastern Atlantic and the Indian Ocean. We found seven out of 11 previously known haplotypes as well as five new haplotypes. One haplotype resembles a new genetic clade, increasing the number of clades from six to seven. Some of these clades seem to be cosmopolitan whereas others appear to be endemic. The phylogeography also shows that different clades occupy different ecological niches and identifies several euryoecious haplotypes with a cosmopolitic distribution as well as some stenoecious haplotypes with an endemic distribution. Haplotypes of different clades differ substantially in their phylogeographic distribution according to latitude. The genetic data also suggest deep phylogenetic branching patterns between clades.     

Caribbean placozoan phylogeography

We here address placozoan distribution and phylogeography in five locations in the Caribbean Sea. We performed a coarse-resolution presence/absence survey of placozoans in Belize, Bermuda, Grenada, Jamaica, and Panama and a fine-resolution study of the distribution of placozoans in Twin Cays, Belize. Placozoans were recovered in every country sampled. Animals were sequenced at the mitochondrial 16S rDNA locus, and our analysis identified four of the five previously identified clades present in the Caribbean. In addition, we discovered two new haplotypes within one of these clades, and we found sympatric clades in Belize, Bermuda, Jamaica, and Panama. These studies provide further molecular evidence for species diversity within the Phylum Placozoa.     

Genetic variation in the stygobitic shrimp Creaseria morleyi (Decapoda: Palaemonidae), evidence of bottlenecks and re‐invasions in the Yucatan Peninsula

The karstic nature of the Yucatan Peninsula promotes the formation of submerged caves and sinkholes that are inhabited by an endemic subterranean water fauna. By contrast with most other micro‐endemic stygobitic species, the freshwater palaemonid shrimp Creaseria morleyi is widely distributed across the northern part of the peninsula. In the present study, we investigated the phylogeographic structure of C. morleyi using two mitochondrial genes as markers, and explored hypotheses related to its evolution in the peninsula. DNA from 14 localities was extracted, and the 16S rRNA and cytochrome oxidase subunit I (COI) genes were amplified and sequenced. The different haplotypes were identified to construct a haplotype network and perform a nested clade analysis. Five haplotypes of the 16S gene were obtained, with a maximum divergence of 0.5%. One of these haplotypes is widely distributed and the most divergent is located in the north‐western section of the peninsula. Twelve haplotypes for the COI gene were found with a maximum divergence of 2%, showing the same spatial pattern. The analysis revealed two significantly different clades corresponding to populations in the centre and south‐east of the peninsula as a consequence of restricted genetic flow with isolation‐by‐distance. The divergence time of these two clades was 40–120 thousand years. The genetic variation in C. morleyi, the relationship between haplotypes and their geographic distribution, along with the geological history of the Yucatan Peninsula, may indicate that this variation is a relict of an ancient marked genetic structure reduced by changes in sea level that resulted in a series of bottlenecks. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 315–325.     

Cytochrome b barcoding, molecular systematics and geographic differentiation in rabbitfishes (Siganidae)

The fish genus Siganus (Siganidae) is widely distributed in the coastal habitats of all the tropical Indo-Pacific, with 28 nominal species recognized so far, based on general morphology and coloration patterns. A mitochondrial phylogeny of 16 Siganidae species, based on the partial nucleotide sequences of the cytochome b gene, was produced. Individual haplotypes of given nominal species generally clustered at the extremity of long branches, thus validating the current taxonomy. However, S. lineatus haplotypes formed a paraphyletic group including S. guttatus, while S. fuscescens haplotypes were apparently splitted in two groups, calling for further investigation. S. woodlandi and S. argenteus formed a monophyletic group, as expected from their close morphological relatedness, although they were separated by a substantial, 14.5-16.3% nucleotide distance. Among eight species sampled from different locations across the Indo-West Pacific, S. argenteus and S. spinus showed the lowest degree of geographic differentiation, a result that correlated well with their extended pelagic larval stage. Fixation index estimates were high in all six other species tested (S. doliatus, S. fuscescens, S. lineatus, S. puellus, S. punctatus, S. vulpinus). The cytochrome b gene fragment chosen here proved useful as a barcode in Siganidae.     

Phylogeography of Arcterica nana (Ericaceae) suggests another range expansion history of Japanese alpine plants

We conducted a phylogeographic study on the alpine plant Arcterica nana based on haplotypes of chloroplast DNA. Using a sequence of approximately 1,071 bp of intergenic spacers of chloroplast DNA (trnT-L, psbB–psbF), we detected 13 haplotypes among 193 individuals sampled from 22 populations. Two dominant haplotypes were distributed over the entire range of this species in Japan, and we found several local private haplotypes. An analysis of molecular variance (AMOVA) indicated no geographic structure within the haplotype distribution. In addition, the genetic distance was not related to its corresponding geographic distance (Mantel test: r=−0.049, P=0.66), indicating a homogeneous geographic structure throughout the entire distribution range in the Japanese archipelago. The most parsimonious explanation for this geographic structure is that A. nana spread across its extant distribution range in the Japanese archipelago through a recent range expansion event. However, this pattern is inconsistent with the previous phylogeography of Japanese alpine plants, which reveals that haplotypes in central Honshu are differentiated from those in more northern regions. Arcterica nana may have experienced a different history from other alpine plants, suggesting that the history of Japanese alpine flora may include at least two different geographic radiation patterns.     

Occurrence in the field of a long-term, year-round, stable population of placozoans

Long-term field studies on placozoans (Trichoplax adhaerens), including both substrate sampling and slide sampling, were carried out at a subtidal site near Shirahama, Japan. Samples of natural substrate materials from the field, such as stones, shells, or fragments of coral, were particularly useful for obtaining placozoans. Results from the substrate sampling indicate that placozoans are present year-round at the study site. Large intermittent peaks in the number of animals collected at the study site occurred roughly once a year, between late summer and the beginning of winter. Placozoans were present every year from 1989 through 2000. A seawater aquarium was also studied and provided a considerable number of placozoans for more than 1 year.     

Phylogeography of lateral plate dimorphism in the freshwater type of ninespine sticklebacks, genus Pungitius

The freshwater type of ninespine sticklebacks, genus Pungitius, is widely distributed in northern Japan and reproductively isolated from other genetically divergent types endemic to small regions in Japan. This type expresses dimorphism in its lateral plate morphology: complete and partial row morphs. The two morphs show a parapatric distribution in Japan. To clarify the process involving the distribution of these two morphs, we examined their phylogeography based on restriction fragment length polymorphism of an entire mitochondrial DNA (mtDNA). The survey was carried out with seven restriction enzymes on the populations of the freshwater type collected from 41 localities across the distribution range in Japan, and 6 further Pungitius populations from the Okhotsk Sea coast of Russia were appended. An unweighted pair-group method with arithmetic averages (UPGMA) tree among 54 mtDNA haplotypes resolved eight clustering groups that differed in sequence divergence by approximately 1.3%–2.1%. Two of the eight groups were found only in Russia. mtDNA phylogenies constructed by neighbor-joining and Wagner parsimony methods suggested that the haplotypes of each plate morph were polyphyletic. The geographic distribution pattern of these groups suggests that they should be classified into two broad categories, one with extensive distribution and the other with localized distribution of the constituent haplotypes within a group. The former groups were found mainly in the populations with the completely plated morph and the latter groups with the partially plated morph. It is supposed that twice dispersals of dimorphic or complete plated ancestors and genetic differentiation during the interglacial played an important role in the formation of the present distribution of the two morphs in Japan. Received: March 28, 2000 / Revised: November 3, 2000 / Accepted: January 16, 2001     

Demographic History of Shorea curtisii (Dipterocarpaceae) Inferred from Chloroplast DNA Sequence Variations

We assessed the variability of chloroplast DNA sequences in populations of the dipterocarp forest tree, Shorea curtisii. This species is widely distributed in hill and coastal hill dipterocarp forests of the Malay Peninsula, whereas isolated populations are found in the coastal hills of north Borneo. Two chloroplast DNA regions (1555 bp of trnH‐psbA‐trnK and 925 bp of trnL‐trnF) were sequenced from 123 individuals collected from six Malay Peninsula and two Bornean populations. There were 15 chloroplast haplotypes derived from 16 polymorphic sites. A haplotype network revealed two distinct haplogroups that correlate with two geographic regions, the Malay Peninsula and Borneo. These two haplogroups differed by a number of mutations, and no haplotypes were shared between populations from the different geographic regions. This suggests an ancient diversification of these haplogroups, and that long‐distance seed dispersal was unlikely to have occurred during the Pleistocene when the Sunda Shelf was a contiguous landmass. Phylogenetic analysis of the haplotypes together with those found in other Shorea species showed that two haplogroups in S. curtisii appear in different positions of the phylogenetic tree. This could be explained by the persistence of ancestral polymorphisms or by ancient chloroplast capture. Low levels of genetic differentiation were found between populations within each geographic region. Signature of a bottleneck followed by demographic expansion was detected in the Malay Peninsula haplogroup. The presence of two distinct evolutionary lineages in the different regions suggests that they should be managed independently to conserve the major sources of genetic diversity in S. curtisii.     

Phylogeography of the Atlanto-Mediterranean sea cucumber Holothuria (Holothuria) mammata: the combined effects of historical processes and current oceanographical pattern

We assessed the genetic structure of populations of the widely distributed sea cucumber Holothuria (Holothuria) mammata Grube, 1840, and investigated the effects of marine barriers to gene flow and historical processes. Several potential genetic breaks were considered, which would separate the Atlantic and Mediterranean basins, the isolated Macaronesian Islands from the other locations analysed, and the Western Mediterranean and Aegean Sea (Eastern Mediterranean). We analysed mitochondrial 16S and COI gene sequences from 177 individuals from four Atlantic locations and four Mediterranean locations. Haplotype diversity was high (H=0.9307 for 16S and 0.9203 for COI), and the haplotypes were closely related (π=0.0058 for 16S and 0.0071 for COI). The lowest genetic diversities were found in the Aegean Sea population. Our results showed that the COI gene was more variable and more useful for the detection of population structure than the 16S gene. The distribution of mtDNA haplotypes, the pairwise F(ST) values and the results of exact tests and amova revealed: (i) a significant genetic break between the population in the Aegean Sea and those in the other locations, as supported by both mitochondrial genes, and (ii) weak differentiation of the Canary and Azores Islands from the other populations; however, the populations from the Macaronesian Islands, Algarve and West Mediterranean could be considered to be a panmictic metapopulation. Isolation by distance was not identified in H. (H.) mammata. Historical events behind the observed findings, together with the current oceanographic patterns, were proposed and discussed as the main factors that determine the population structure and genetic signature of H. (H.) mammata.     

Field biology of placozoans (Trichoplax): distribution, diversity, biotic interactions

The goal of this review is to highlight what little is known,and point to the bulk of what is yet to be learned, about thenatural history of placozoans in the field—in order tostimulate a broader search for placozoans and a fuller explorationof their distribution, diversity, and all other aspects of theirenigmatic lives. The documented geographic distribution of placozoanslies mostly in the nearshore, warm, marine waters of the tropicsand subtropics. Although placozoans have long been viewed asbenthic organisms, they can be more readily collected from thewater column, well above the sea bottom. The full life-historyof placozoans is unknown, including the nature of this abundantpelagic phase and all details of sexual reproduction and development.We note observations on the biota associated with placozoansin field collections, in particular the other regular membersof the microcommunity in which placozoans occur on our collectingplates and on some factors influencing this assemblage. Amongthe animals found are some potential predators against whichplacozoans appear to be defended, although the mechanisms arestill to be examined. Also yet to be uncovered is the full breadthof diversity in this phylum, certainly underrepresented by itssingle named species. We report here greatly expanded distributionsfor known haplotypes and fresh specimens that include a newhaplotype, and we review the evidence that many more almostcertainly await discovery. We also describe some methods forcollecting and handling these small, fragile animals.     

Genetic and acoustic population structuring in the Okinawa least horseshoe bat: are intercolony acoustic differences maintained by vertical maternal transmission?

The origin and meaning of echolocation call frequency variation within rhinolophid bats is not well understood despite an increasing number of allopatric and sympatric examples being documented. A bimodal distribution of mean regional call frequency within the Okinawa‐jima Island population of Rhinolophus cornutus pumilus (Rhinolophidae) provided a unique opportunity to investigate geographic call frequency variation early in its development. Individual resting echolocation frequencies, partial mitochondrial DNA D‐loop sequences and genotypes from six microsatellite loci were obtained from 288 individuals in 11 colonies across the entire length of the island, and nearby Kume‐jima Island. Acoustic differences (5–8 kHz) observed between the north and south regions have been maintained despite evidence of sufficient nuclear gene flow across the middle of the island. Significant subdivision of maternally inherited D‐loop haplotypes suggested a limitation of movement of females between regions, but not within the regions, and was evidence of female philopatry. These results support a ‘maternal transmission’ hypothesis whereby the difference in the constant frequency (CF) component between the regions is maintained by mother–offspring transmission of CF, the restricted dispersal of females between regions and small effective population size. We suggest that the mean 5–8 kHz call frequency difference between the regions might develop through random cultural drift.     

Phylogeography of Asparagopsis taxiformis revisited: Combined mtDNA data provide novel insights into population structure in Japan

Six intraspecific lineages (Lineages 1–6) of Asparagopsis taxiformis have been previously established based on mitochondrial cox2‐cox3 intergenic spacer and a partial cox1 sequences. ‘Lineage 2’ (L2) was suggested to be a recent introduction to the Mediterranean Sea, but its source population has not yet been identified. In order to clarify the nature of northwestern Pacific populations, we performed extensive sampling in Japan (60 individuals from 16 locations) and molecular phylogenetic analyses based on mitochondrial sequences. Sixteen additional individuals, collected from eight locations in the Indo‐Pacific, Caribbean, and Mediterranean regions, were also analyzed. Combined sequence analyses revealed that the Japanese populations only consisted of L2. Out of 19 combined haplotypes identified within L2, two are shared between Japan and the Mediterranean Sea and the Hawaiian Islands, and 12 were identified as endemic to Japan. Genetic analyses of population differentiation suggested that Japanese populations are genetically isolated from the Mediterranean and the Hawaiian populations. A genetic disjunction appears to separate two subpopulations within Japan: one between Toi and Kagoshima and the other between Ojikajima Island and Kagoshima in the Kyushu area.     

Diversity and Geographical Distribution of Phytoplasmas infecting China-tree in Argentina

Yellows diseases associated with phytoplasmas cause high mortality in China‐tree (Melia azedarach) in Argentina, but there has been no previous large‐scale survey to determine their diversity and geographical distribution. To assess the presence and identity of phytoplasmas affecting this species throughout the country, 425 samples of symptomatic trees collected at different geographic locations were analysed by a polymerase chain reaction (using universal and group‐specific primers) and restriction fragment length polymorphism. Phytoplasmas belonging to 16SrIII‐B group were detected at almost every location sampled, whereas 16SrXIII‐C group phytoplasmas, reported for the first time in Argentina, were only found in two regions sharing similar agro‐ecological characteristics (Northeast provinces and Tucumán). Double infections with 16SrIII‐B and 16SrXIII‐C group phytoplasmas were also recorded. Nucleotide sequencing of the 16S rDNA of three Argentinian 16SrXIII‐C group phytoplasma isolates revealed high identity (99.6–99.3%) with the CbY1 isolate reported from Bolivia.     

Genetic population structure of Japanese river sculpin Cottus pollux (Cottidae) large-egg type,inferred from mitochondrial DNA sequences

The geographic distribution of mitochondrial DNA haplotypes from Japanese river sculpin Cottus pollux large-egg type (LE), collected from 55 locations in 22 rivers over most of the species’ range in Japan, was examined to assess for patterns of population genetic structure. The 87 haplotypes observed from C. pollux LE were distinguishable from C. pollux middle-egg type and small-egg type haplotypes from previously published research. Cottus pollux LE from each river were largely represented by diagnostic mtDNA haplotypes, with limited geographical associations of haplotypes, suggesting that each river must be treated as a separate management unit.     

Geographic patterns of genetic differentiation within the restricted range of the endangered Stephens' kangaroo rat Dipodomys stephensi

Using mtDNA variation in the kangaroo rat Dipodomys stephensi, we found no support for the hypothesis that a species with an historically restricted range will exhibit low levels of genetic polymorphism and little genetic structure. Dipodomys stephensi has long been restricted to a few interior coastal valleys in southern California encompassing an area of approximately 70 x 40 km; however, we found high levels of genetic variation over much of its range and significant genetic structure both within and between regions. We also found evidence for a recent range expansion. Dipodomys stephensi is a federally endangered species that is separated from D. panamintinus, its presumed sister taxon, by a mountain range to the north. We assessed genetic variation by sequencing 645 bases of the mitochondrial d-loop from 61 individuals sampled from 16 locations across the species range and rooted their relationship using two D. panamintinus individuals. Despite its limited geographic range, the level of mtDNA variation in D. stephensi is comparable to that of other rodents, including that of the more widely distributed D. panamintinus. This variation revealed significant regional differentiation. The northern, central, and southern regions of the range differ in both the level and the distribution of genetic variation. Phylogenetic analysis revealed that the center of the range contains the most diversity of lineages, including the most basal. In this region and in the north, most haplotypes were found at only a single location (25/29), or at a pair of nearby locations (3/29). In addition, related haplotypes clustered geographically. These results are consistent with long-term demographic stability characterized by limited dispersal and high local effective population size. Further support for this conclusion is the finding of unique diversity in two northern peripheral populations, Norco and Potrero Creek (PC). However, in sharp contrast, one haplotype (CC) was found at five of 11 central and northern locations and comprised 18% of individuals sampled. The atypical distribution of the CC haplotype reflected a pattern seen more strongly in the southern region. Here the CC haplotype comprised 69% of the sample and was found at all five sampling locations. Consequently, the southern region had very low genetic variability. We propose that this dominance of CC was probably due to a local population bottleneck that occurred during a recent range expansion into the southern region.     

Genetic diversity of nuclear and mitochondrial genomes in Pinus parviflora Sieb. & Zucc. (Pinaceae) populations

Genetic diversities of the nuclear and mitochondrial genomes in Pinus parviflora were studied in 16 populations, which were distributed across most of the species' range in Japan. Six mitochondrial DNA haplotypes were identified among the 16 populations. The intrapopulation diversity of allozymes was similar to that of other endemic woody species (H(S)=0.259). Although P. parviflora is distributed in discrete populations, differentiation between these was very low (G(ST)=0.044). In addition, the extent of genetic differentiation between two varieties (var. pentaphylla and var. parviflora) was extremely low (G(VT)=0.001). Intrapopulation diversity of mitochondrial DNA was also very low (H(S)=0.098), but population differentiation was high (G(ST)=0.863). Moreover, the distribution of haplotypes reflected the taxonomic differences between P. parviflora var. pentaphylla and var. parviflora. The populations of var. pentaphylla and var. parviflora contained different haplotypes. Differing modes of inheritance may account for the differences in nuclear and mitochondrial genetic diversity.     

Phylogeography of Arabis serrata (Brassicaceae) in the Japanese Archipelago based on chloroplast DNA variations

Arabis serrata (Brassicaceae), a perennial plant widely distributed along the Japanese Archipelago, occurs in various habitats: for example, limestone zones, serpentine barrens, volcanic soils, and roadsides. It likely survived by adapting to its surrounding environment, resulting in great morphological and ecological variation. In this study, we performed a phylogeographic analysis to examine past changes in the distribution of A. serrata following climate oscillations during the Pleistocene. To cover most of A. serrata's range, leaves were collected from eight to ten individuals randomly selected from each of 37 populations in the Japanese Archipelago. Two chloroplast noncoding regions of the samples were amplified and sequenced: trnT(GGU)‐psbD and trnH(GUG)‐psbA spacers. Twenty‐five haplotypes were detected and distinguished by 31 substitutions. Four main haplotypes were observed in many populations distributed throughout the Japanese Archipelago. According to the genetic boundaries detected using the Monmonier algorithm, A. serrata is clustered into four groups, each including several populations: Hokkaido Island, northern mainland Honshu, central Japan, and western Japan. The boundaries, however, were not robust because all genetic parameters did not support the differentiation among groups. These results indicate the absence of an obvious geographic structure in the distribution of A. serrata, suggesting that this species has experienced a rapid range expansion in postglacial times.     

Comparison of three organelle markers for phylogeographic inference in Batrachospermum helminthosum (Batrachospermales, Rhodophyta) from North America

Phylogeographic signal provided by the newly developed 23S plastid rRNA marker (universal plastid amplicon, UPA) and the cytochrome oxidase subunit I gene marker (COI) in the freshwater red alga Batrachospermum helminthosum, throughout its range in North America, was investigated. These markers were compared in individuals from a previous study using the cytochrome oxidase 2–3 spacer region ( cox 2–3), which has yielded the most useful data to date with 13 haplotypes among geographic locations. Five haplotypes were resolved for the UPA, differing by only one to two base pairs (bp), and we conclude that this marker may be more appropriate for studying interspecific variation. In contrast, the COI gene revealed 16 haplotypes, differing from one to 44 base pairs or up to 6.6% sequence variation. The intraspecific variation of COI in this taxon is much greater than that reported thus far for marine red algae (generally <5 bp). The intraspecific variation within B. helminthosum is in accord with levels shown in Batrachospermum macrosporum (48 bp within distant locations in Brazil). The COI gene is comparable to the cox 2–3 spacer for phylogeographic studies as the haplotype networks were similar and showed the same geographic patterns. To our knowledge, this is the first comparison of these three regions for phylogeographic research in the red algae.     

Population Structure of the Lower Keys Marsh Rabbit as Determined by Mitochondrial DNA Analysis

ABSTRACT We used nucleotide sequence data from a mitochondrial DNA fragment to characterize variation within the endangered Lower Keys marsh rabbit (Sylvilagus palustris hefneri). We observed 5 unique mitochondrial haplotypes across different sampling sites in the Lower Florida Keys, USA. Based on the frequency of these haplotypes at different geographic locations and relationships among haplotypes, we observed 2 distinct clades or groups of sampling sites (western and eastern clades). These 2 groups showed low levels of gene flow. Regardless of their origin, marsh rabbits from the Lower Florida Keys can be separated into 2 genetically distinct management units, which should be considered prior to implementation of translocations as a means of offsetting recent population declines.     

Phylogeny of the Mexican coastal leopard frogs of the Rana berlandieri group based on mtDNA sequences

Phylogenetic relationships among specimens from 25 different locations for the six Mexican coastal leopard frog species of the Rana berlandieri species group were investigated using 797 bp of the mitochondrial 12S rDNA gene. Relationships among the haplotypes obtained were recovered using maximum parsimony and Bayesian analyses. Most of the clades recovered by both tree building methods are strongly supported, but conflicting clades recovered by each analysis are generally poorly supported. Both analyses reject the previously proposed subgroupings of the R. berlandieri species group. Based on the strongly supported relationships, genetic differentiation, and geographic distribution of the haplotypes examined, nine independent lineages appear to comprise the group of study. However, confirmation of the new proposed lineages will require further analyses based on other genetic markers and additional samples that cover their entire geographic distribution. Concordance was noted between Miocene-Pliocene geological and climatic events in Mexico and the relationships recovered among the lineages proposed and their geographic distribution.