Hemocyanin gene family evolution in spiders (Araneae), with implications for phylogenetic relationships and divergence times in the infraorder Mygalomorphae

Hemocyanins are multimeric copper-containing hemolymph proteins involved in oxygen binding and transport in all major arthropod lineages. Most arachnids have seven primary subunits (encoded by paralogous genes a–g), which combine to form a 24-mer (4 × 6) quaternary structure. Within some spider lineages, however, hemocyanin evolution has been a dynamic process with extensive paralog duplication and loss. We have obtained hemocyanin gene sequences from numerous representatives of the spider infraorders Mygalomorphae and Araneomorphae in order to infer the evolution of the hemocyanin gene family and estimate spider relationships using these conserved loci. Our hemocyanin gene tree is largely consistent with the previous hypotheses of paralog relationships based on immunological studies, but reveals some discrepancies in which paralog types have been lost or duplicated in specific spider lineages. Analyses of concatenated hemocyanin sequences resolved deep nodes in the spider phylogeny and recovered a number of clades that are supported by other molecular studies, particularly for mygalomorph taxa. The concatenated data set is also used to estimate dates of higher-level spider divergences and suggests that the diversification of extant mygalomorphs preceded that of extant araneomorphs. Spiders are diverse in behavior and respiratory morphology, and our results are beneficial for comparative analyses of spider respiration. Lastly, the conserved hemocyanin sequences allow for the inference of spider relationships and ancient divergence dates.     

Species delimitation and phylogeography of Aphonopelma hentzi (Araneae, Mygalomorphae, Theraphosidae): cryptic diversity in North American tarantulas

Background

The primary objective of this study is to reconstruct the phylogeny of the hentzi species group and sister species in the North American tarantula genus, Aphonopelma, using a set of mitochondrial DNA markers that include the animal “barcoding gene”. An mtDNA genealogy is used to consider questions regarding species boundary delimitation and to evaluate timing of divergence to infer historical biogeographic events that played a role in shaping the present-day diversity and distribution. We aimed to identify potential refugial locations, directionality of range expansion, and test whether A. hentzi post-glacial expansion fit a predicted time frame.

Methods and Findings

A Bayesian phylogenetic approach was used to analyze a 2051 base pair (bp) mtDNA data matrix comprising aligned fragments of the gene regions CO1 (1165 bp) and ND1-16S (886 bp). Multiple species delimitation techniques (DNA tree-based methods, a “barcode gap” using percent of pairwise sequence divergence (uncorrected p-distances), and the GMYC method) consistently recognized a number of divergent and genealogically exclusive groups.

Conclusions

The use of numerous species delimitation methods, in concert, provide an effective approach to dissecting species boundaries in this spider group; as well they seem to provide strong evidence for a number of nominal, previously undiscovered, and cryptic species. Our data also indicate that Pleistocene habitat fragmentation and subsequent range expansion events may have shaped contemporary phylogeographic patterns of Aphonopelma diversity in the southwestern United States, particularly for the A. hentzi species group. These findings indicate that future species delimitation approaches need to be analyzed in context of a number of factors, such as the sampling distribution, loci used, biogeographic history, breadth of morphological variation, ecological factors, and behavioral data, to make truly integrative decisions about what constitutes an evolutionary lineage recognized as a “species”.     

The elongation factor-1delta (EF-1delta) originates from gene duplication of an EF-1beta ancestor and fusion with a protein-binding domain.

The molecular evolution of two components of elongation factor-1 (EF-1), EF-1beta and EF-1delta was analysed using the distance matrix, the maximum parsimony and the maximum likelihood methods, after careful alignment of protein and cDNA sequences. The topology of the phylogenetic trees obtained supports monophyly of plant EF-1beta and EF-1beta' sequences, and monophyly of higher eukaryotic animal EF-1beta and EF-1delta sequences. EF-1beta and EF-1delta are homologous in their C-terminal domain. EF-1delta, which emerged before arthropods, originates from a beta-type ancestor gene and fusion with a leucine zipper N-terminal motif. Plant EF-1beta and EF-1beta' correspond to paralogous genes whose ancestor was most likely duplicated before the emergence of monocotyledons and dicotyledons.     

Phylogenetic utility and evidence for multiple copies of elongation factor-1alpha in the spider genus Habronattus (Araneae: Salticidae)

In the continuing quest for informative genes for use in molecular systematics, the protein-coding gene Elongation factor-1alpha (EF-1alpha) has rapidly become one of the most prevalent "single-copy" nuclear genes utilized, particularly in arthropods. This paper explores the molecular evolutionary dynamics and phylogenetic utility of EF-1alpha in the salticid spider genus Habronattus. As has been reported for other arthropod lineages, our studies indicate that multiple (two) copies of EF-1alpha exist in Habronattus. These copies differ in intron structure and thus in size, making it possible to easily separate PCR amplification products. We present data for an intronless EF-1alpha copy for three Habronattus species. The presence of nonsense mutations and generally elevated rates of amino acid change suggest that this copy is evolving under relaxed functional constraints in Habronattus. A larger taxon sample (50 species plus outgroups) is presented for an EF-1alpha copy that includes both intron and exon regions. Characteristics of both regions suggest that this is a functional, orthologous copy in the species sampled. Maximum-likelihood relative-rate comparisons show that exon third codon sites are evolving more than 100 times as fast as second codon sites in these sequences and that intron sites are evolving about twice as fast as exon third sites. In combination, the EF-1alpha data provide robust, species-level phylogenetic signal that is largely congruent with morphologically well supported areas of Habronattus phylogeny. The recovery of some novel clades, and the unexpected fragmentation of others, suggests areas requiring further phylogenetic attention.     

Phylogenetic reconsideration of Myrmekiaphila systematics with a description of the new trapdoor spider species Myrmekiaphila tigris (Araneae, Mygalomorphae, Cyrtaucheniidae, Euctenizinae) from Auburn, Alabama

The trapdoor spider genus Myrmekiaphila currently comprises 11 nominal species. A recent molecular phylogenetic evaluation of the group identified a number of problems with respect to how species and species groups were delineated by Bond and Platnick in their 2007 taxonomic revision of the genus. We report herein the discovery of a new species, Myrmekiaphila tigrissp. n. The phylogenetic position of the species is evaluated using a molecular phylogenetic approach based on a set of mtDNA markers. Our preferred phylogenetic hypothesis supports the recognition of a new species and further highlights the need to more carefully investigate species boundaries within the genus. These results further indicate that palpal bulb morphology is rapidly evolving and has likely been a contributing factor in rendering a number of species paraphyletic with respect to the molecular data.     

The reproductive period of tarantulas is constrained by their thermal preferences (Araneae,Theraphosidae)

Locomotor and physiological performance of ectotherms are affected by temperature. Thermoregulation is achieved by changes in behavior and the selection of micro-habitats with adequate temperatures to maintain the body temperature (T_b) within a range of preference. Apart from this temperature dependence at spatial scales, ectotherms are also affected by temperature at temporal scale. For instance, ectotherms can only be active some months of the year, particularly in temperate environments. Tarantulas are ectotherms that live in burrows most of their life. Nevertheless, after the sexual maturation molt, males leave their refugia and start a wandering life searching for females to mate. The reproductive period varies among species. In some species walking males are seen in late spring or early summer, while in other species males are only seen during fall or winter. Apart from the differences in lifestyles after maturation, tarantulas exhibit sexual dimorphisms in longevity and body mass, having smaller, shorter-lived males. Thus, to optimize energetic budgets, decreasing thermoregulation costs, we hypothesize and examine a putative correlation between an individual's preferred body temperature (T_pref) and the environmental temperature during the reproductive period. Hence, we characterize T_pref in seven tarantula species and analyze which factors (i.e., time of day, body mass, and sex) correlated with it. Furthermore, we assess putative correlated evolution of T_pref with ambient temperature (minima, mean, and maxima) during the reproductive period by means of phylogenetic independent contrasts. We did not find differences in thermal preferences between sexes; and only one species, Acanthoscurria suina, exhibited diel differences in T_pref. We found evidence of correlated evolution between T_pref and minimum temperature during the reproductive period among all seven species studied herein. Our results show that the reproductive period is constrained by thermal preferences, dictating when males can start their wandering life to mate.     

The ultrastructure of the book lungs of the Italian trap-door spider Cteniza sp. (Araneae,Mygalomorphae, Ctenizidae)

The fine structure of book lungs is not homogeneous across Arachnids and is considered phylogenetically informative, however few reports on the ultrastructural features of this organ have been published. In this study, we examined the general morphology and ultrastructure of adult spiders of the genus Cteniza. The respiratory system of Cteniza sp. consists of two pairs of well-developed book lungs, which is considered indicative of primitive spiders. The general organization of the book lungs is similar to that described for other arachnids and consists of leaves of alternating air and hemolymph channels. The air channels are lined with cuticle and open to an atrium that leads to a slit-like spiracle. The air channels are held open by cuticular trabeculae. The space holders in the hemolymph channels are pillar trabeculae formed by two cells from the opposed walls. The pillar cells have a complex ultrastructure that includes an interdigitating connection, gap junctions, microtubules and hemidesmosomes. These features apparently help strengthen the pillar cells and their interconnections with each other and the underlying cuticle. The cytoskeleton resembles that of arthropod tendon cells where substantial structural support is needed.     

Phylogenetic relationships of the spider family Tetragnathidae (Araneae, Araneoidea) based on morphological and DNA sequence data

The monophyly of Tetragnathidae including the species composition of the family (e.g., Are Nephila and their relatives part of this lineage?), the phylogenetic relationships of its various lineages, and the exact placement of Tetragnathidae within Araneoidea have been three recalcitrant problems in spider systematics. Most studies on tetragnathid phylogeny have focused on morphological and behavioral data, but little molecular work has been published to date. To address these issues we combine previous morphological and behavioral data with novel molecular data including nuclear ribosomal RNA genes 18S and 28S, mitochondrial ribosomal RNA genes 12S and 16S and protein‐coding genes from the mitochondrion [cytochrome c oxidase subunit I (COI)] and from the nucleus (histone H3), totaling ca. 6.3 kb of sequence data per taxon. These data were analyzed using direct optimization and static homology using both parsimony and Bayesian methods. Our results indicate monophyly of Tetragnathidae, Tetragnathinae, Leucauginae, the “Nanometa clade” and the subfamily Metainae, which, with the exception of the later subfamily, received high nodal support. Morphological synapomorphies that support these clades are also discussed. The position of tetragnathids with respect to the rest of the araneoid spiders remains largely unresolved but tetragnathids and nephilids were never recovered as sister taxa. The combined dataset suggests that Nephilidae is sister to Araneidae; furthermore, the sister group of Nephila is the clade composed by Herennia plus Nephilengys and this pattern has clear implications for understanding the comparative biology of the group. Tetragnathidae is most likely sister to some members of the “reduced piriform clade” and nephilids constitute the most‐basal lineage of araneids.     

Transitive predatory relationships of spider species (Arachnida, Araneae) in laboratory tests

Interspecific predation of spiders was studied in the laboratory in view of possible competition in the wild. Certain species killed other species even if handicapped by smaller size. Thirty eight spider species were involved in such a relationship and their predatory relationships were significantly reliable and transitive ('linear' or 'non-triangular'). A theridiid species (Theridion tinctum) showed the highest rank in terms of killing seven 'beta species', i.e. species capable of killing at least one alien species of larger size than themselves. Another theridiid (Steatoda grossa) obtained the second rank by killing five beta species. Experiments in both the wild and laboratory may, further, investigate other factors than body size that may be relevant to competition, such as behaviour-related characteristics (e.g. web structure and biting speed) and ecological factors (e.g. different susceptibilities of the species to parasite or predator attack).     

Theraphosidae phylogeny: relationships of the ‘Ischnocolinae’ genera (Araneae,Mygalomorphae)

The mygalomorph spider subfamily ‘Ischnocolinae’ was originally established as a group based on the presence of divided tarsal scopula. Later, the divided condition of the scopula was considered the plesiomorphic state, which could not support the monophyly of ‘Ischnocolinae’. In Raven 1985, the subfamily was considered paraphyletic, pending a phylogenetic analysis to reinvestigate monophyletic groups. This study comprises such a phylogenetic analysis, based on morphological data, that includes representatives of all genera currently included in ‘Ischnocolinae’ as well as representatives of all other nine Theraphosidae subfamilies (Thrigmopoeinae, Ornithoctoninae, Eumenophorinae, Stromatopelminae, Harpactirinae, Selenogyrinae, Theraphosinae, Aviculariinae and Selenocosmiinae). The family Theraphosidae is considered monophyletic and expanded to include three additional genera previously considered as possible Barychelidae, namely Brachionopus (as Harpactirinae), Trichopelma and Reichlingia (as Ischnocolinae sensu stricto) while ‘Ischnocolinae’ as previously defined does not appear as monophyletic. However, two monophyletic groups were defined as subfamilies to include some former ‘Ischnocolinae’ representatives. The first group includes Acanthopelma rufescens, Trichopelma nitidum, Reichlingia annae, Ischnocolus spp., Holothele rondoni, Holothele culebrae and Holothele aff culebrae and is hereby named as Ischnocolinae (sensu stricto). The other subgroup comprises Sickius longibulbi, Holothele incei, Holothele aff incei, Guyruita spp., Schismatothele lineata, Hemiercus modestus, Holothele colonica and Holothele sp., together established as Schismatothelinae subfam. nov. Several genera included in former ‘Ischnocolinae’ appear as monophyletic (Catumiri, Oligoxystre, Heterothele, Nesiergus, Chaetopelma, Ischnocolus, Guyruita and Plesiophrictus). However, the genera Holothele, Schismatothele and Hemiercus deserve more attention in order to evaluate their intrarelationships and inclusion of species.     

A phylogenetic analysis of the orb-weaving spider family Araneidae (Arachnida, Araneae)

We present the first cladistic analysis focused at the tribal and subfamily level of the orb-weaving spider family Araneidae. The data matrix of 82 characters scored for 57 arancid genera of 6 subfamilies and 19 tribes (and 13 genera from 8 outgroup families) resulted in 16 slightly different, most parsimonious trees. Successive weighting corroborated 62 of the 66 informative nodes on these cladograms; one is recommended as the 'working' araneid phylogcny. The sister group of Araneidae is all other Araneoidea. Araneidae comprises two major clades: the subfamily Araneinae, and the 'argiopoid' clade, which includes all other subfamilies and most tribes (((Gasteracanthinae, Caerostreae), (((Micratheninae, Xylcthreae), Eruyosaccus ), (Eurycorminae, Arciinae)), Cyrlarachninae), ((Argiopinae, Cyrtophorinae), Arachnureae)). Cyrtarachneae and Mastophoreae are united in a new subfamily, Cyr-tarachninae. The spiny orb-weavers alone (Gasteracanthinae and Micratheninae) are not monophyletic. The mimetid subfamily Arciinae and the 'tetragnathid' genus Zygiella are araneids, but .Nephila (and other tetragnathids) are not. On the preferred tree, web decorations (stabilimenta) evolved 9 times within 15 genera, and were lost once. The use of silk to subdue prey evolved once in cribellate and four times in ecribillate orb weavers. Sexual size dimorphism evolved once in nephilines, twice in araneids, and reverted to monomorphism five times. Evolution in other genitalic and somatic characters is also assessed; behavioral and spinneret features arc most consistent (male genitalia, leg and prosomal features least consistent) on the phylogeny.     

从核rDNA序列探讨隙蛛亚科Coelotinae的分类地位

隙蛛亚科Coelotinae主要分布于东亚地区,其中我国的已有种类占到全世界种数的一半以上,因此对于我国隙蛛类蜘蛛的研究已经成为世界暗蛛科研究的重点之一。隙蛛亚科属于无筛器类群,于1893年,由Cambridge以隙蛛属为模式属而建立,归属于无筛器的漏斗蛛科。之后,虽然经历了数次修订     

In situ assessment of mRNA accessibility in heterogeneous tissue samples using elongation factor-1α (EF-1α)

 Elongation factor-1α (EF-1α) is an evolutionarily highly conserved universal cofactor of protein synthesis in all living cells. In this study, its use as a positive control in situ hybridization assays for specific detection of mRNA sequences was evaluated. Northern blot analysis of various non-neoplastic and neoplastic cultured cells of different stages of confluence, cell shape, and cell cycle status revealed that EF-1α had a lower and more homogeneous expression than did β-actin. In situ hybridization assays using digoxigenin-labeled riboprobes for the detection of EF-1α mRNA in routinely formalin-fixed, paraffin-embedded tissue sections showed that EF-1α is a suitable positive control in all types of cells. However, variation of protease pretreatments demonstrated distinct and sometimes mutually exclusive digestion conditions for different cell types within the same tissue sample. Our results indicate that detection of EF-1α mRNA is an appropriate internal standard for in situ hybridization assays and that it is useful to control artifacts such as false negatives caused by inappropriate protease pretreatments. The observed variability of optimal protease pretreatments for different cell types within the same tissue section strengthens the importance of a positive control in in situ hybridization assays. Accepted: 17 December 1996     

Phylogeny and taxonomy of thegenera of south-western North American Euctenizinae trapdoor spidersand their relatives (Araneae: Mygalomorphae, Cyrtaucheniidae)

The primary focus of this paper is to evaluate the monophylyand intergeneric relationships of the cyrtaucheniid subfamily Euctenizinaeand to a lesser degree the monophyly of Cyrtaucheniidae. Using 71morphological characters scored for 29 mygalomorph taxa our cladisticanalysis shows that Cyrtaucheniidae is likely paraphyletic withrespect to the Domiothelina, the clade that comprises the Migidae,Actinopodidae, Ctenizidae, and Idiopidae. Together, the Domiothelinaand Cyrtaucheniidae have been treated as the Rastelloidina clade.A strict interpretation of rastelloid classification based on ourcladogram would require the establishment of four additional spiderfamilies. However, we choose to use informal names for these cladesso that these taxa can be validated by subsequent studies of mygalomorphphylogeny before formal names are introduced. The phylogenetic analysisalso serves as a vehicle for examining the patterns of homoplasyobserved in mygalomorphs. The secondary focus of this paper is ataxonomic revision of Euctenizinae genera from the south-westernUnited States that includes a key to its genera. The cyrtaucheniid genera Enrico and Astrosoga areconsidered junior synonyms of Eucteniza. Actinoxia and Nemesoides arejunior ­synonyms of Aptostichus . At present theNorth American Euctenizinae comprises these seven nominal genera: Eucteniza, Neoapachella gen.nov. ( Neoapachella rothi sp. nov. ), Myrmekiaphila,Entychides, Promyrmekiaphila, Apomastus gen. nov. ( Apomastusschlingeri sp. nov. ), and Aptostichus .  © 2002The Linnean Society of London . Zoological Journal of the LinneanSociety, 2002, 136 , 487−534     

Four new spider species of the family Theridiosomatidae (Arachnida,Araneae) from caves in Laos

Four new species of the spider family Theridiosomatidae are described from caves in Laos: Alaria cavernicolasp. n. (♂♀), A. navicularissp. n., (♂♀) A. bicornissp. n. (♂♀), Chthonopes thakekensissp. n. (♀). Diagnoses and illustrations for all new taxa are given. All holotypes are deposited in the Senckenberg Research Institute in Frankfurt am Main, Germany (SMF).