An evolutionary timescale for terrestrial isopods and a lack of molecular support for the monophyly of Oniscidea (Crustacea: Isopoda)

The marine metazoan fauna first diversified in the early Cambrian, but terrestrial environments were not colonized until at least 100 million years later. Among the groups of organisms that successfully colonized land is the crustacean order Isopoda. Of the 10,000 described isopod species, ~ 3,600 species from the suborder Oniscidea are terrestrial. Although it is widely thought that isopods colonized land only once, some studies have failed to confirm the monophyly of Oniscidea. To infer the evolutionary relationships among isopod lineages, we conducted phylogenetic analyses of nuclear 18S and 28S and mitochondrial COI genes using maximum-likelihood and Bayesian methods. We also analyzed a second data set comprising all of the mitochondrial protein-coding genes from a smaller sample of isopod taxa. Based on our analyses using a relaxed molecular clock, we dated the origin of terrestrial isopods at 289.5 million years ago (95% credibility interval 219.6–358.9 million years ago). These predate the known fossil record of these taxa and coincide with the formation of the supercontinent Pangaea and with the diversification of vascular plants on land. Our results suggest that the terrestrial environment has been colonized more than once by isopods. The monophyly of the suborder Oniscidea was not supported in any of our analyses, conflicting with classical views based on morphology. This draws attention to the need for further work on this group of isopods.     

Exploring phylogenetic informativeness and nuclear copies of mitochondrial DNA (numts) in three commonly used mitochondrial genes: mitochondrial phylogeny of peppermint,cleaner, and semi‐terrestrial shrimps (Caridea: Lysmata,Exhippolysmata, and Merguia)

Of paramount importance to studies that profit from molecular trees is the accuracy and robustness of the reconstructed phylogenies. Causes of systematic error that can mislead phylogenetic methods include nuclear copies of mitochondrial DNA (numts) and low phylogenetic informativeness (PI). Herein, numts and PI were explored in three mitochondrial genes commonly used for phylogenetic reconstruction: 16S, 12S, and cytochrome c oxidase I (COI). Shrimps from the genera Lysmata, Exhippolysmata, and Merguia were used as a model system. The existence of: (1) multiple bands on gels of COI and 12S polymerase chain reaction (PCR) products from various species; (2) double peaks, background noise, and ambiguity in sequence chromatograms of COI and 12S PCR products that produced a single clear band in other species; and (3) indels, stop codons, and considerable composition bias in COI‐like cloned sequences of one problematic species (Lysmata seticaudata), was interpreted as evidence of pervasive non‐functional nuclear copies of mitochondrial DNA (numts) of the targeted COI (and probably 12S) mtDNA fragment. The information content of the three mtDNA markers studied was investigated using PI profiling, spectral analysis, and neighbour‐nets. Marker‐specific PI profiles suggested that the COI marker has the highest information content and greatest power for resolving both shallow and deep nodes in trees depicting the phylogenetic relationship among the species studied. Nonetheless, spectral analysis of splits and neighbour‐nets suggested that the 16S and 12S markers were equally or even more powerful than the COI marker for resolving nodes at all phylogenetic levels. Altogether, these analyses suggest that all three mtDNA markers are equally useful for resolving phylogenetic relationships in the shrimps studied, and that PI profiling is not necessarily useful to estimate overall gene utility. A ‘total‐evidence’ phylogenetic analysis that included 34 species and used a concatenated data set of 1403 characters (from reliable 16S, 12S and COI sequences), demonstrated that the genus Lysmata is paraphyletic, and that the monophyletic clade comprising species of Lysmata and Exhippolysmata can be divided into four well‐supported subclades (Neotropical, Cleaner, Cosmopolitan, and Morphovariable). © 2013 The Linnean Society of London     

A molecular phylogeny of the checkered beetles and a description of Epiclininae a new subfamily (Coleoptera: Cleroidea: Cleridae)

We provide the first molecular phylogeny of the clerid lineage (Coleoptera: Cleridae, Thanerocleridae) within the superfamily Cleroidea to examine the two most recently proposed hypotheses of higher level classification. Phylogenetic relationships of checkered beetles were inferred from approximately ～5000 nt of both nuclear and mitochondrial rDNA (28S, 16S and 12S) and the mitochondrial protein‐coding gene COI. A worldwide sample of ～70 genera representing almost a quarter of generic diversity of the clerid lineage was included and phylogenies were reconstructed using Bayesian and Maximum Likelihood approaches. Results support the monophyly of many proposed subfamilies but were not entirely congruent with either current classification system. The subfamilial relationships within the Cleridae are resolved with support for three main lineages. Tillinae are supported as the sister group to all other subfamilies within the Cleridae, whereas Thaneroclerinae, Korynetinae and a new subfamily formally described here, Epiclininae subf.n ., form a sister group to Clerinae + Hydnocerinae.     

On the importance of fine‐scale sampling in detecting alpha taxonomic diversity among saproxylic invertebrates: A velvet worm (Onychophora: Opisthopatus amaxhosa) template

The phylogeography and social structure of the narrow endemic velvet worm species Opisthopatus amaxhosa were investigated by conducting fine‐scale sampling in its distribution range in the Eastern Cape province of South Africa. In addition, and as part of larger grant on forest biodiversity, Opisthopatus specimens sampled at localities not included during a recent evaluation of the genus were included in a new phylogeny. A total of 89 specimens from 18 sample localities were collected at three forest patches for O. amaxhosa samples, while an additional six Opisthopatus sample localities were included. For O. amaxhosa, we sequenced the COI locus for all specimens, while a subset of specimens was sequenced for two nuclear loci, 18S rRNA and the fushi tarazu intron (FTz). Phylogenetic analyses using maximum likelihood and Bayesian inferences of the latter species revealed the presence of two highly divergent clades, characterised by marked uncorrected sequence divergence values. In addition, these two clades did not share any maternal haplotypes, were characterised by high F_ST values and fixed nuclear difference for the 18S rRNA locus, while the FTz intron was genetically invariant. Furthermore, the application of scanning electron microscopy between the two genetically divergent clades also revealed the presence of fixed ventral and dorsal scale numbers. Collectively, this provides evidence for a novel species that is present at a fine scale. Divergence time estimations suggest that the two clades diverged during the late and early Pleistocene with climatic cycling potentially causal to the fragmentation. The social structure was male‐biased, and samples from the same logs were not always genetically identical. At the broader scale, the inclusion of new specimens within Opisthopatus revealed no novel lineages. Fine‐scale sampling appears more important to detect alpha taxonomic diversity compared to broadscale sampling.     

Complete mitochondrial genome of the wild silkmoth,Saturnia boisduvalii (Lepidoptera: Saturniidae)

We sequenced mitochondrial genome (mitogenome) of the wild silkmoth, Saturnia boisduvalii (Lepidoptera: Saturniidae), which occurs in mainland Korea, and compared it with other species in Bombycoidea to characterize the genomic evolution of the superfamily. We found that the composition and arrangement of genes in the 15,257‐bp S. boisduvalii genome are typical of the majority of Lepidoptera, and the genome is biased toward A/T nucleotides, as previously reported. Comparison of individual gene divergence among bombycoid species showed that ND6 was most variable (p‐distance = 0.21), whereas COI and COII were most conserved, indicating that of all the protein‐coding genes (PCGs) ND6 appear to have evolved most rapidly. Thus, other PCGs beside COI are potential alternative markers, where scrutinized discrimination among species is required.     

Landscape‐scale genetic differentiation of a mycangial fungus associated with the ambrosia beetle,Xylosandrus germanus (Blandford) (Curculionidae:Scolytinae) in Japan

In this study, we examined the genetic structures of the ambrosia fungus isolated from mycangia of the scolytine beetle, Xylosandrus germanus to understand their co‐evolutionary relationships. We analyzed datasets of three ambrosia fungus loci (18S rDNA, 28S rDNA, and the β‐tubulin gene) and a X. germanus locus dataset (cytochrome c oxidase subunit 1 (COI) mitochondrial DNA). The ambrosia fungi were separated into three cultural morphptypes, and their haplotypes were distinguished by phylogenetic analysis on the basis of the three loci. The COI phylogenetic analysis revealed three distinct genetic lineages (clades A, B, and C) within X. germanus, each of which corresponded to specific ambrosia fungus cultural morphptypes. The fungal symbiont phylogeny was not concordant with that of the beetle. Our results suggest that X. germanus may be unable to exchange its mycangial fungi, but extraordinary horizontal transmission of symbiotic fungi between the beetle's lineages occurred at least once during the evolutionary history of this symbiosis.     

Amplicon pyrosequencing late Pleistocene permafrost: the removal of putative contaminant sequences and small‐scale reproducibility

DNA sequencing of ancient permafrost samples can be used to reconstruct past plant, animal and bacterial communities. In this study, we assess the small‐scale reproducibility of taxonomic composition obtained from sequencing four molecular markers (mitochondrial 12S ribosomal DNA (rDNA), prokaryote 16S rDNA, mitochondrial cox1 and chloroplast trnL intron) from two soil cores sampled 10 cm apart. In addition, sequenced control reactions were used to produce a contaminant library that was used to filter similar sequences from sample libraries. Contaminant filtering resulted in the removal of 1% of reads or 0.3% of operational taxonomic units. We found similar richness, overlap, abundance and taxonomic diversity from the 12S, 16S and trnL markers from each soil core. Jaccard dissimilarity across the two soil cores was highest for metazoan taxa detected by the 12S and cox1 markers. Taxonomic community distances were similar for each marker across the two soil cores when the chi‐squared metric was used; however, the 12S and cox1 markers did not cluster well when the Goodall similarity metric was used. A comparison of plant macrofossil vs. read abundance corroborates previous work that suggests eastern Beringia was dominated by grasses and forbs during cold stages of the Pleistocene, a habitat that is restricted to isolated sites in the present‐day Yukon.     

Unmasking alpha diversity,cladogenesis and biogeographical patterning in an ancient panarthropod lineage (Onychophora: Peripatopsidae: Opisthopatus cinctipes) with the description of five novel species

Speciation and biogeographical patterning in the velvet worm Opisthopatus cinctipes was examined under a null hypothesis that numerous discrete lineages are nested within the species. A total of 184 O. cinctipes specimens, together with a single specimen of each of the two congeneric point endemic sister species (O. roseus and O. herbertorum), were collected throughout the forest archipelago in the Eastern Cape, KwaZulu‐Natal and Mpumalanga provinces of South Africa. All specimens were sequenced for two partial mitochondrial DNA loci (COI and 12S rRNA), while a single specimen from each locality was sequenced for the nuclear 18S rRNA locus. Evolutionary relationships were assessed using maximum‐likelihood and Bayesian inferences, while divergence time estimations were conducted using BEAST. A Bayesian species delimitation approach was undertaken to explore the number of possible novel lineages nested within Opisthopatus, while population genetic structure was examined for the COI locus using ARLEQUIN. Phylogenetic results revealed that O. cinctipes is a species complex comprising seven geographically discrete and statistically well‐supported clades. An independent statistical approach to species delimitations circumscribed ca. 67 species. Results from divergence time estimation and rate constancy tests revealed near constant net diversification occurring throughout the Eocene and Oligocene with subdivision of ranges during the Miocene. Gross morphological characters such as leg pair number within O. cinctipes were invariant, while dorsal and ventral integument colour was highly polymorphic. However, scanning electron microscopy revealed considerable differences both between and within clades. The caveats associated with both morphological and algorithmic delineation of species boundaries are discussed. The five novel Opisthopatus species are described.     

Molecular phylogeography of Troglophilus cave crickets (Orthoptera,Rhaphidophoridae): A combination of vicariance and dispersal drove diversification in the East Mediterranean region

In this study, we investigated the molecular phylogenetic divergence and historical biogeography of cave crickets belonging to the genus Troglophilus (Orthoptera, Rhaphidophoridae) from caves in eastern Mediterranean and Anatolia regions. Three mitochondrial DNA genes (COI, 12S rDNA, and 16S rDNA) and two nuclear ones (18S rDNA and 28S rDNA) were amplified and partially sequenced to reconstruct phylogenetic relationships among most of the known Troglophilus species. Results showed a well‐resolved phylogeny with three main clades representing the Balkan, the Anatolian, and the Cycladian–Cretan lineages. Based on Bayesian analyses, we applied a relaxed molecular clock model to estimate the divergence times between these three lineages. Dating estimates indicate that radiation of the ingroup might have been triggered by the opening of the Mid‐Aegean trench, while the uplift of the Anatolian Plateau in Turkey and the changes of relief, emergence, and disappearance of orographic and hydrographical barriers in the Balkan Peninsula are potential paleogeographic events responsible for the initial diversification of the genus Troglophilus. A possible biogeographic scenario, reconstructed using S‐DIVA with RASP software, suggested that the current distribution of Troglophilus species can be explained by a combination of both dispersal and vicariance events that occurred in particular in the ancestral populations. The radiation of Troglophilus species likely started from the Aegean and proceeded eastward to Anatolia and westward to the Balkan region. Results are additionally compared to those available for Dolichopoda, the only other representative genus of Rhaphidophoridae present in the Mediterranean area.     

Relationships within the Munnopsidae (Crustacea, Isopoda, Asellota) based on three genes

The Munnopsidae are a diverse group of asellote isopods that are an important component of deep‐sea fauna. Morphologically‐based phylogenetic inference attempts have proven to be of limited use due to the ecological and morphological diversity within the clade. Monophyly of the family is well‐established but relationships within the group remain unresolved. This project is the first molecularly‐based effort focused specifically on resolving phylogenetic relationships within the Munnopsidae. Partial 28S and COI and complete 18S genes were sequenced for 28 asellotes, 15 additional taxa were included from which only one or two of the three target sequences could be obtained, and 18S sequences for five additional taxa were available from GenBank. Sequences were analysed both as individual genes and in combination using Bayesian and maximum parsimony approaches. Each gene provided a phylogenetic signal that could be identified in the combined analyses, with 18S analyses providing the most resolution of phylogenetic relationships. The available representatives of subfamilies Munnopsinae and Ilyarachninae were monophyletic, as was the genus Munneurycope. Relationships within the subfamily Munnopsinae were well‐resolved by thorough taxon sampling, several new species were placed, and the need for taxonomic revision of Munnopsis/Munnopsoides was supported. These analyses supported putative Eurycope paraphyly and emphasized the need for careful revision of this highly variable genus. Tytthocope was sister to Munnopsurus. Syneurycope was suggested as the sister group to the ilyarachnines. Combined analyses provided increased support for clades suggested in at least two individual gene analyses and for clades not strongly contradicted by individual analyses. Further work is required to fully resolve the munnopsid phylogeny and should consist of increased taxon sampling for the complete 18S sequence and possibly identification of at least one slowly evolving, nuclear protein‐coding gene to resolve the basal polytomy and enable placement of the root.     

The Halomonhystera disjuncta population is homogeneous across the Håkon Mosby mud volcano (Barents Sea) but is genetically differentiated from its shallow‐water relatives

The deep sea has a high biodiversity and a characteristic bathyal fauna. Earlier evidence suggested that at least some shallow‐water species invaded the ecosystem followed by radiation leading to endemic deep‐sea lineages with a genetic and/or morphological similarity to their shallow‐water counterparts. The nematode Halomonhystera disjuncta has been reported from shallow‐water habitats and the deep sea [Håkon Mosby mud volcano (HMMV)], but the morphological features and the phylogenetic relationships between deep‐sea and shallow‐water representatives remain largely unknown. Furthermore, nothing is known about the genetic structure of the H. disjuncta population within the HMMV. This study is the first integrative approach in which the morphological and phylogenetic relationships between a deep‐sea and shallow‐water free‐living nematode species are investigated. To elucidate the phylogenetic relationships, we analysed the mitochondrial gene Cytochrome oxidase c subunit I (COI) and three nuclear ribosomal genes (Internal Transcribed Spacer region, 18S and the D2D3 region of 28S). Our results show that deep‐sea nematodes comprise an endemic lineage compared to the shallow‐water representatives with different morphometric features. COI genetic divergence between the deep‐sea and shallow‐water specimens ranges between 19.1% and 25.2%. Taking these findings into account, we conclude that the deep‐sea form is a new species. amova revealed no genetic structure across the HMMV, suggesting that nematodes are able to disperse efficiently in the mud volcano.     

Phylogenetic analysis based on multiple gene sequences revealing cryptic biodiversity in Simulium multistriatum group (Diptera: Simuliidae) in Thailand

Phylogenetic relationships among four species of the Simulium multistriatum group (Diptera: Simuliidae) in Thailand were examined based on two mitochondrial genes (COI, COII) and one nuclear gene (18S/ITS1). Simulium takense was found to be genetically divergent (>20.3% for COI) from the other species, consistent with their distinctive morphology. Simulium chainarongi and S. chaliowae were monophyletic but were included in paraphyletic S. fenestratum. Simulium fenestratum was divided into three distinct lineages with high levels of genetic divergence. This suggests that S. fenestratum is a species complex. Neither morphological nor cytological examinations revealed evidence of sibling species. The clades derived from phylogenetic analyses were found to be correlated with the ecological conditions of larval habitat. Therefore, ecological adaptation may have played a role in black fly diversification and evolution. These results suggest the use of integrated, multidisciplinary approaches for fully understanding black fly biodiversity and systematics.     

Molecular phylogeny of Omaliinae (Coleoptera: Staphylinidae) and its implications for evolution of atypically long elytra in rove beetles

Staphylinidae, or rove beetles, are a megadiverse family known for their typically very short elytra exposing most of the abdomen, but the putatively early-derived subfamily Omaliinae and its relatives have been known to include multiple taxa with unusually long elytra. The ancestral elytral length of the family and of this subfamily have long been debated. We present a phylogenetic analysis of Omaliinae based on partial mitochondrial COI (1488 bp), COII (366 bp), 12S rDNA (353 bp), nuclear 18S rDNA (1814 bp), 28S rDNA (876 bp) and CAD (869 bp) data. In all, 51 species in 31 genera and four outgroup species were included. The concatenated sequences were analysed by both parsimony- and model-based (Bayesian and maximum likelihood) methods. The subfamily Omaliinae was not supported as a monophyletic group. The model-based analyses (Bayesian and maximum likelihood trees) showed Empelinae nested within Omaliinae (excluding Corneolabiini), whereas parsimony analysis found all three putative ingroup subfamilies, Empelinae, Glypholomatinae and Microsilphinae, grouped within Omaliinae. Within the Omaliinae, the tribes Coryphiini and Eusphalerini were each supported as monophyletic, whereas Anthophagini and Omaliini were each nonmonophyletic. We hypothesize that there have been at least four independent origins of long elytra from short elytra in the omaliine lineage.     

Analysis of mitochondrial COI sequences of the Diachasmimorpha longicaudata (Hymenoptera: Braconidae) species complex in Thailand

Diachasmimorpha longicaudata is an Opiinae parasitoid used to control tephritid fruit flies, which cause tremendous economic losses of fruits worldwide. In Thailand, D. longicaudata is classified as three sibling species, DLA, DLB and DLBB, based on the morphological and biological species concepts but their genetic variation has not been studied. Therefore, we investigated the genetic differentiation of the mitochondrial COI gene to clarify the ambiguous taxonomy of this species complex. The 603‐bp COI region was sequenced from laboratory‐bred colonies and field‐collected specimens from seven locations representing five geographical regions in Thailand. DLA was associated with the host Bactrocera correcta while DLB and DLBB were associated with Bactrocera dorsalis. The interspecific nucleotide differences of COI sequences among the three groups ranged from 6.70% to 7.62% (Kimura 2‐parameter distance), which adequately separates species complexes within the order Hymenoptera and supports the current sibling species classification. The neighbor joining, maximum likelihood and consensus Bayesian phylogenetic trees constructed from COI sequences revealed that the three sibling species of laboratory and field‐collected D. longicaudata are monophyletic with 100% support. The high genetic variation and molecular phylogeny of the COI sequences were shown to discriminate between the D. longicaudata species examined in this study.     

Hidden diversity of Ctenophora revealed by new mitochondrial COI primers and sequences

The mitochondrial gene cytochrome-c-oxidase subunit 1 (COI) is useful in many taxa for phylogenetics, population genetics, metabarcoding, and rapid species identifications. However, the phylum Ctenophora (comb jellies) has historically been difficult to study due to divergent mitochondrial sequences and the corresponding inability to amplify COI with degenerate and standard COI “barcoding” primers. As a result, there are very few COI sequences available for ctenophores, despite over 200 described species in the phylum. Here, we designed new primers and amplified the COI fragment from members of all major groups of ctenophores, including many undescribed species. Phylogenetic analyses of the resulting COI sequences revealed high diversity within many groups that was not evident from more conserved 18S rDNA sequences, in particular among the Lobata (Ctenophora; Tentaculata; Lobata). The COI phylogenetic results also revealed unexpected community structure within the genus Bolinopsis, suggested new species within the genus Bathocyroe, and supported the ecological and morphological differences of some species such as Lampocteis cruentiventer and similar undescribed lobates (Lampocteis sp. “V” stratified by depth, and “A” differentiated by colour). The newly designed primers reported herein provide important tools to enable researchers to illuminate the diversity of ctenophores worldwide via quick molecular identifications, improve the ability to analyse environmental DNA by improving reference libraries and amplifications, and enable a new breadth of population genetic studies.     

Phylogeny of cockroaches (Insecta,Dictyoptera, Blattodea), with placement of aberrant taxa and exploration of out‐group sampling

We addressed the phylogeny of cockroaches using DNA sequence data from a broad taxon sample of Dictyoptera and other non‐endopterygotan insect orders. We paid special attention to several taxa in which relationships are controversial, or where no molecular evidence has been used previously: Nocticolidae, a family of small, often cave‐dwelling cockroaches, has been suggested to be the sister group of the predaceous Mantodea or of the cockroach family Polyphagidae; Lamproblatta, traditionally placed in Blattidae, has recently been given family status and placed as sister to Polyphagidae; and Saltoblattella montistabularis Bohn, Picker, Klass & Colville, a jumping cockroach, which has not yet been included in any phylogenetic studies. We used mitochondrial (COI + COII and 16S) and nuclear (18S and 28S) genes, and analysed the data using Bayesian inference (BI) and maximum likelihood (ML). Nocticolidae was recovered as sister to Polyphagidae. Lamproblatta was recovered as sister to Blattidae, consistent with the traditional placement (not based on phylogenetic analysis). However, because of the limited support for this relationship and conflict with earlier morphology‐based phylogenetic hypotheses, we retain Lamproblattidae. S. montistabularis was consistently placed as sister to Ectobius sylvestris Poda (Blaberoidea: Ectobinae), indicating that the saltatorial hindlegs of this genus are a relatively recent adaptation. Isoptera was placed within Blattodea as sister to Cryptocercidae. Nocticolidae + Polyphagidae was sister to Isoptera + Cryptocercidae, and Blaberoidea was sister to the remaining Blattodea.     

Molecular phylogenetic and divergence time estimation analyses of the sawfly subfamily Selandriinae (Hymenoptera: Tenthredinidae)

Selandriinae, a subfamily of family Tenthredinidae (Hymenoptera: Symphyta), comprises multiple tribes, each of which has a relationship with specific plant group. The host specificity of the Selandriinae taxa provides a good model to examine the coevolution between hosts and insects. However, few phylogenetic studies for the Selandriinae obscure the evolutionary scenario with their host‐plants. The present study is a molecular phylogenetic analysis of 19 selandriine species based on mitochondrial genes (12S: 461 sites, 16S: 262sites and COI: 495 sites) and nuclear genes (18S: 773 sites and 28S: 495 sites). The results suggested three of six studied tribes are genetically isolated. Moreover, estimation of the time of molecular divergence showed that the Selandriinae clearly diverged at the same time as their host‐plants (monocots and ferns). These results suggested that the Selandriinae species might have codiversified with their hosts.     

Nuclear insertions and heteroplasmy of mitochondrial DNA as two sources of intra‐individual genomic variation in grasshoppers

We examined the level of intra‐individual variation in a region of the mitochondrial genome coding for cytochrome oxydase 1 (COI) in two grasshopper species using a clone‐and‐sequence analysis of hundreds of sequences. In both Locusta migratoria and Chortoicetes terminifera, we found that 60–65% of the clones were unique COI‐like sequences. Among these COI‐like sequences, 70–75% diverged by less than 1% from the real mitochondrial haplotypes, and were likely to represent microheteroplasmic molecules. About 20% of the COI‐like sequences diverged by more than 9% from the mitochondrial haplotypes, and generally included stop codons, suggesting that these sequences were nuclear mitochondrial pseudogenes (NUMTs). Only six sequences, diverging by 2–6% from the mitochondrial haplotypes, were identified as potentially misleading in phylogenetic studies. In addition, we found that five sequences from C. terminifera were associated with mobile elements or repetitive DNA families.     

Assessing the Biodiversity of Monoraphidium Using 18S rDNA Sequences

The taxonomy of the genus Monoraphidium is unclear due in part to the absence of morphological features to clearly distinguish one species from another. Phytoplankton samples collected from lakes in the Arrowwood National Refuge in eastern North Dakota were found to contain several morphological species of Monoraphidium. Eighteen Monoraphidium isolates were examined with light microscopy and six morphological species were identified. PCR–RFLP of the 18S rDNA was used to type the isolates. Following digestion by Hae III and Taq I, the 18S rDNA PCR–RFLP patterns indicated 10 different types. Presently, the 18S rDNA product is being sequenced for each of the 10 types. By examining morphological characters and 18S rDNA sequences, congruence between morphology and sequence data may be compared. Also, because there is a lack of morphological characters defining Monoraphidium species, diversity within the 18S rDNA sequences may aid in the taxonomy of the genus and its place within the Chlorococcales. Supported by National Science Foundation Grants MCB‐0084188 and DBI‐0070387.     

Phylogeographic patterns of a lower intertidal isopod in the Gulf of California and the Caribbean and comparison with other intertidal isopods

A growing body of knowledge on the diversity and evolution of intertidal isopods across different regions worldwide has enhanced our understanding on biological diversification at the poorly studied, yet vast, sea–land interface. High genetic divergences among numerous allopatric lineages have been identified within presumed single broadly distributed species. Excirolana mayana is an intertidal isopod that is commonly found in sandy beaches throughout the Gulf of California. Its distribution in the Pacific extends from this basin to Colombia and in the Atlantic from Florida to Venezuela. Despite its broad distribution and ecological importance, its evolutionary history has been largely neglected. Herein, we examined phylogeographic patterns of E. mayana in the Gulf of California and the Caribbean, based on maximum‐likelihood and Bayesian phylogenetic analyses of DNA sequences from four mitochondrial genes (16S rDNA, 12S rDNA, cytochrome oxidase I gene, and cytochrome b gene). We compared the phylogeographic patterns of E. mayana with those of the coastal isopods Ligia and Excirolana braziliensis (Gulf of California and Caribbean) and Tylos (Gulf of California). We found highly divergent lineages in both, the Gulf of California and Caribbean, suggesting the presence of multiple species. We identified two instances of Atlantic–Pacific divergences. Some geographical structuring among the major clades found in the Caribbean is observed. Haplotypes from the Gulf of California form a monophyletic group sister to a lineage found in Venezuela. Phylogeographic patterns of E. mayana in the Gulf of California differ from those observed in Ligia and Tylos in this region. Nonetheless, several clades of E. mayana have similar distributions to clades of these two other isopod taxa. The high levels of cryptic diversity detected in E. mayana also pose challenges for the conservation of this isopod and its fragile environment, the sandy shores.