Homogeneous Lineage Research Articles

Morphological and Genetic Differentiation of Loliolus (Nipponololigo) beka (Cephalopoda: Loliginidae) in Coastal China

The population genetic structure of 211 samples of Loliolus (Nipponololigo) beka, which were selected from across seven geographic localities—in the Bohai Sea, the Yellow Sea and the East China Sea—were analyzed using mitochondrial COI and 16S rRNA gene markers. Phylogenetic trees and a haplotype network both showed that the L. (N.) beka localities were genetically distinct, forming two homogeneous lineages: Lineage A and Lineage B. The results of an AMOVA showed that the genetic variation in the L. (N.) beka populations was dominated by the genetic variation between the two lineages, and both the genetic distance and genetic differentiation indices indicated that the genetic differentiation between the two lineages of L. (N.) beka in Chinese waters had reached the level of species divergence. To further confirm the differences between the two lineages shown in the molecular results, we performed a detailed analysis based on morphometric observations and a multivariate statistical analysis to compare the morphology characteristics of Lineage A and Lineage B. The results showed that there were significant differences (p < 0.05) in the ventral mantle length (VML); the mantle width index (MWI); the fin width index (FWI); the head length index (HLI); the left Arm IV length index (LALI4), the right Arm III length index (RALI3), the right Arm IV length index (RALI4), and the hectocotylized proportion of the left Arm IV length (HcL%) between the two lineages. The differences between the two lineages were also supported by the analysis results for the number of sucker ring teeth. Accordingly, the results of the morphological analysis further confirmed the molecular analysis and provided additional evidence for the presence of the cryptic species of L. (N.) beka in the coastal areas of China.

Multilocus sequence typing of phytoplasmas associated with Flavescence dorée disease in Tuscany vineyards identifies a highly homogeneous lineage in the subgroup 16SrV–C

Flavescence dorée (FD) is the most threatening grapevine yellows (GY) disease in Europe. Despite strict control measures, alarming signs of the spread of the disease in viticultural areas continue to be detected. FD is attributed to infection by phytoplasma strains of an incidentally cited species, ‘Candidatus Phytoplasma vitis’. In 2017, a GY field survey was carried out in traditional viticulture areas of Tuscany, central Italy. FD phytoplasma (FDp) was detected in 85 GY symptomatic vines, accounting for 17% of a total of 500 symptomatic samples screened. The FDp-positive vines were scattered in 50 vineyards across seven Tuscan provinces, indicating the distribution of FDp has further extended to central and southwestern parts of Tuscany including Florence and Livorno. Multilocus sequence typing of 15 representative FDp strains from six affected vineyards revealed that the Tuscan FDp strains constitute a highly homogeneous lineage within the subgroup 16SrV–C (FD-C). Single nucleotide polymorphisms (SNPs) were identified in the 16S rRNA, rp, and secY genes of the Tuscan FDp lineage. Such SNP markers provide clues to understanding the genetic relationships among different FDp lineages present in Europe and are useful for searching potential vectors and reservoirs involved in the spread of the FDp in the Tuscan region.

The Genus Pachyma (Syn. Wolfiporia) Reinstated and Species Clarification of the Cultivated Medicinal Mushroom "Fuling" in China.

The fungus “Fuling” has been used in Chinese traditional medicine for more than 2000 years, and its sclerotia have a wide range of biological activities including antitumour, immunomodulation, anti-inflammation, antioxidation, anti-aging etc. This prized medicinal mushroom also known as “Hoelen” is resurrected from a piece of pre-Linnean scientific literature. Fries treated it as Pachyma hoelen Fr. and mentioned that it was cultivated on pine trees in China. However, this name had been almost forgotten, and Poria cocos (syn. Wolfiporia cocos), originally described from North America, and known as “Tuckahoe” has been applied to “Fuling” in most publications. Although Merrill mentioned a 100 years ago that Asian Pachyma hoelen and North American P. cocos are similar but different, no comprehensive taxonomical studies have been carried out on the East Asian Pachyma hoelen and its related species. Based on phylogenetic analyses and morphological examination on both the sclerotia and the basidiocarps which are very seldomly developed, the East Asian samples of Pachyma hoelen including sclerotia, commercial strains for cultivation and fruiting bodies, nested in a strongly supported, homogeneous lineage which clearly separated from the lineages of North American Wolfiporia cocos and other species. So we confirm that the widely cultivated “Fuling” Pachyma hoelen in East Asia is not conspecific with the North American Wolfiporia cocos. Based on the changes in Art. 59 of the International Code of Nomenclature for algae, fungi, and plants, the generic name Pachyma, which was sanctioned by Fries, has nomenclatural priority (ICN, Art. F.3.1), and this name well represents the economically important stage of the generic type. So we propose to use Pachyma rather than Wolfiporia, and subsequently Pachyma hoelen and Pachyma cocos are the valid names for “Fuling” in East Asia and “Tuckahoe” in North America, respectively. In addition, a new combination, Pachyma pseudococos, is proposed. Furthermore, it seems that Pachyma cocos is a species complex, and that three species exist in North America.

Contrasting phylogeographic patterns of earthworms (Crassiclitellata, Lumbricidae) on near-shore mediterranean islands

Comparative phylogeography is a powerful methodological approach to understand the particular evolutionary phenomena that occur in islands. This method has been rarely applied to insular earthworm communities. These soil animals show a striking dichotomy in their phylogeographic patterns: deeply divergent, regionally structured linages and widely distributed, genetically homogeneous lineages. An intensive earthworm sampling campaign in the Southern France archipelago of Hyères served as an opportunity to check for the existence of these patterns in the framework of a near-shore, continental archipelago. Molecular barcoding (COI sequencing) was performed for earthworm communities of 31 localities, and the seven with more informative distributions were chosen for phylogenetic inference and genetic diversity evaluation. A time-calibrated phylogeny was obtained to estimate a temporal framework for colonization and divergence within the islands, and haplotype networks were used to visualize relationships between populations in detail. Four species (Allolobophora chlorotica, Aporrectodea nocturna, Scherotheca cf. dugesi and Scherotheca rhodana) showed clades restricted to the archipelago and hints of Plio-Pleistocene in situ divergence. The other three species (Aporrectodea rosea, Aporrectodea trapezoides and Eiseniella tetraedra) lacked geographic structure, being nested in clades with individuals from distant countries; their arrival to the archipelago appeared consistent with human-mediated introduction. These contrasting phylogeographic patterns are discussed in the context of paleogeographic events affecting the Mediterranean in the Late Tertiary-Quaternary and biological traits of earthworms, with biparental sexual vs parthenogenetic reproduction as the main factor explaining them.

Multilocus phylogeny defines a new classification of Staphylininae (Coleoptera, Staphylinidae), a rove beetle group with high lineage diversity

AbstractWe provide the first multilocus molecular phylogeny of a group corresponding to the former subfamily Staphylininae. Results are corroborated by the morphological, biogeographical and palaeobiological evidence to serve as a baseline for an updated suprageneric classification. The former subfamily Staphylininae is proven to be a lineage sister to the monophyletic Paederinae and reclassified according to a robust phylogeny resolving a number of long‐standing controversies. The subfamily Xantholininae (revised status) is reinstated to contain the tribes Xantholinini, Othiini, Maorothiini and Diochini. Subfamily Platyprosopinae (revised status) is reinstated for the tribes Platyprosopinini, Arrowinini and †Thayeralinini. For a highly peculiar genus Coomania Cameron, formerly in Diochini, a new subfamily Coomaniinae subfam.n. is established and the composition of Diochini (revised status) is changed accordingly. The subfamily Staphylininae (revised status) is reduced to contain the former tribe Staphylinini only. Elevating this mega‐diverse tribe to the subfamily rank opened up an opportunity for its more fractional classification by raising several subtribes to the tribal level as follows: Acylophorini, Afroquediini, Amblyopinini, Antimerini, †Baltognathini, Cyrtoquediini, Erichsoniini, Hyptiomini, Indoquediini, Quediini and Tanygnathinini (revised status for all). As a result, the most species‐rich tribe Staphylinini (revised status) is reduced to the more homogeneous lineage containing the subtribes Algonina, Anisolinina, Philonthina, Philothalpina, Staphylinina and Xanthopygina. Morphological synapomorphies and diagnostic characters supporting all newly defined higher taxa are provided.This published work has been registered on ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:DED8B042‐83C9‐4D10‐B0CB‐B50372B067A9.

Evidence for rapid adaptive evolution of tolerance to chemical treatments in Phytophthora species and its practical implications.

Chemical treatments are used widely in agricultural and natural settings to protect plants from diseases; however, they may exert an important selection pressure on plant pathogens, promoting the development of tolerant isolates through adaptive evolution. Phosphite is used to manage diseases caused by Phytophthora species which include a large number of the most economically damaging plant pathogens worldwide. Phosphite controls the growth of Phytophthora species in planta without killing it; as a result, isolates can develop tolerance to phosphite after prolonged exposure. We investigated the inter- and intra-specific variability in phosphite tolerance of eleven Phytophthora species, including P. ramorum, an internationally important, highly regulated pathogen. Phytophthora ramorum is a good model system because it is comprised of multiple genetically homogeneous lineages. Seven species were found to be consistently sensitive to phosphite based on the low Effective Concentration (EC) 50 values of all isolates tested (amount of phosphite required to inhibit mycelial growth by 50% relative to growth in the absence of phosphite). However, P. ramorum, P. lateralis, P. crassamura and P. cambivora showed intraspecific variability in sensitivity to phosphite, with at least one isolate showing significantly higher tolerance than the other isolates. Within the three P. ramorum evolutionarily divergent lineages tested, NA1 was the most susceptible to phosphite, the NA1 and EU1 lineages showed intralineage variability and the NA2 lineage showed a decreased sensitivity to phosphite overall as all isolates were relatively tolerant. This finding is relevant because NA1 is dominant in the wild and can be controlled using phosphite, while the EU1 lineage has recently been identified in the wild and is phosphite-tolerant, making the treatment approach potentially less effective. Phytophthora ramorum, P. lateralis and P. crassamura are either selfing, homothallic species, or are known to reproduce exclusively clonally, indicating tolerance to phosphite can emerge even in the absence of sexual recombination.

Phylogeography of the widespread creek chub Semotilus atromaculatus (Cypriniformes: Leuciscidae).

The extent and nature of genetic differentiation in Semotilus atromaculatus, one of the most abundant and widespread leuciscids in North America, were evaluated based on mitochondrial (mt) and nuclear DNA sequence variation. Phylogenetic relationships were first inferred based on a fragment of the cytochrome b (cytb) region and the nuclear intron s7 gene for S. atromaculatus and all other congeners as well as representative species from all other genera in the creek chub-plagopterin clade. The phylogeography of major haplogroups of S. atromaculatus was also assessed according to variation in a fragment of the mitochondrial cytb region from 567 individuals across its range. All analyses identified S. thoreauianus, S. lumbee and S. corporalis as reciprocally monophyletic groups. Analyses of nuclear sequence variation resolved S. atromaculatus as a single clade, where S. thoreauianus and S. lumbee were recovered as the sister group to S. atromaculatus, and S. corporalis was resolved as sister to all other species in the genus. Analyses of mtDNA sequence variation recovered S. atromaculatus as three well supported and differentiated monophyletic groups, with a widespread genetically homogeneous lineage extending across most of the current range of the species; a more geographically restricted and geographically structured lineage in the southern Appalachians, sister group to S. lumbee; and a geographically restricted lineage was identified from two Gulf Slope basins. Evidence of complex mito-nuclear discordance and phylogeographic differentiation within S. atromaculatus illustrates that further analysis of widespread species is warranted to understand North American freshwater fish diversity and distributions.

High intra-host Cherry virus A population heterogeneity in cherry trees in Slovakia

Despite the absence of symptoms, next generation sequencing (NGS) of total RNAs from a wild cherry tree revealed a multiple virus infection. Besides prune dwarf virus (PDV) and little cherry virus 2 (LChV-2) the analysis revealed the presence of two genetically divergent cherry virus A (CVA) variants. Their complete genomes could be reconstituted, differing by 14% at the nucleotide level. The presence of these CVA variants in the original cherry tree was confirmed during three consecutive growing seasons by variant-specific RTPCR. The development of a polyvalent RT-PCR assay targeting the 3´ part of the CVA genome allowed the identification of the virus in several other cultivated, wild or ornamental cherries in Slovakia, and the analysis of CVA genomic variability in the amplified region. This has allowed regrouping of known CVA isolates into six major phylogenetic groups, some of which are only recently reported, further extending the known diversity of this virus. In addition, evidence was obtained supporting the recombinant nature of two Slovak isolates and for the presence of “non-cherry” group isolates of CVA in several cherry host plants. Further investigation of the intra-tree CVA variability in five unrelated cherry trees revealed a homogenous lineage in two trees (average nucleotide diversity 0.2-0.5%), while the CVA population in the other trees was composed of variants belonging to three to four different evolutionary lineages (average diversity 7.4-8.8%). The frequent presence of highly divergent molecular variants within the CVA population in single trees further highlights the complex and heterogeneous nature of viral populations infecting perennial hosts.

Interleukin-17 in atherosclerosis and cardiovascular disease: the good, the bad, and the unknown

This editorial refers to ‘Circulating levels of interleukin-17 and cardiovascular outcomes in patients with acute myocardial infarction’, by T. Simon et al. , doi:10.1093/eurheartj/ehs263 Over the past few years our understanding of the importance of adaptive immunity in acute coronary syndromes (ACS) has considerably increased.1 Profound abnormalities have been observed in specific subsets of CD4+T cells, including type 1 helper T (Th1) cells,2,3 CD4+CD28nullT cells,4 and naturally occurring regulatory T cells (Treg cells).5 Recently a new lineage of CD4+T cells, type 17 helper T (Th17) cells producing the signature cytokines interleukin (IL)-17, IL-21, and IL-22, has been identified.6 This has fundamentally changed the Th1/Th2 dichotomy paradigm. The Th1 cell subset is characterized by interferon (IFN)-γ production and T-bet expression, whereas the Th2 cell lineage shows IL-4, IL-5, and IL-10 production and GATA-3 expression. At the other extreme, Treg cells expressing FoxP3 are essential to maintain the homeostasis of cell subsets involved in adaptive immunity by contact-dependent suppression or by releasing anti-inflammatory cytokines, IL-10 and transforming growth factor (TGF)-β1.7 Like Th1, Th2, and Treg cells, specific polarizing cytokines—TGF-β, IL-6, and IL-23—are required for differentiation of Th17 cells which express the transcription factors retinoic acid orphan receptor (ROR) α and RORγt. However, Th17 cells do not represent a homogeneous lineage, as they can be reprogrammed to other T-cell subsets depending on the cytokine environment.6 Accordingly, IL-17 can be co-expressed with a variety of other cytokines including IFN-γ, IL-10, and IL-4. Indeed, a mixed Th1–Th17 subgroup expressing both T-bet and RORγt has been recently identified in the setting of autoimmunity.6 Plasticity …

Finnish Campylobacter jejuni strains of multilocus sequence type ST-22 complex have two lineages with different characteristics.

Background Campylobacter jejuni is the major cause of human bacterial gastroenteritis worldwide, and in a minority of cases, post-infectious complications may occur. ST-22 complex (usually Penner serotype 19) strains have been overrepresented among patients with postinfectious complications of campylobacteriosis. We here present a characterization of a collection of 27 Finnish C. jejuni strains of ST-22 complex, from humans (22 strains) and animal sources (five strains), with the aim of contributing to our knowledge of the pathogenesis of C. jejuni infections.Methodology/Principal FindingsAll strains were analyzed by pulsed-field gel electrophoresis (PFGE) genotyping, lipo-oligosaccharide (LOS) locus class, Y-glutamyl transpeptidase (GGT) activity, in vitro biofilm formation ability, invasion and adhesion in HeLa cells and induction of IL-8 production. ST-22 complex contained five STs (ST-22; ST-1947; ST-1966; ST-3892; ST-3996) which were homogeneous in having sialylated LOS class A1 but on the other hand were distinguished into two major lineages according to the major STs (ST-22 and ST-1947) by different PFGE genotypes and certain other characteristics. All ST-22 strains had similar SmaI PFGE profiles, were GGT positive, and formed biofilms, except one strain, while ST-1947 strains were all GGT negative, did not form biofilm, had significantly higher motility than ST-22 (p<0.05) and had their SmaI PFGE profile. Invasion and adhesion as well as induction of IL-8 production on HeLa cells were strain-dependent characteristics.Conclusions/SignificanceST-22 complex strains, reveal potential for molecular mimicry in host interactions upon infection as they all express sialylated LOS class A1. The two major STs, ST-22 and ST-1947 formed two homogeneous lineages, which differed from each other both phenotypically and genetically, suggesting that the strains may have evolved separately, perhaps by interacting with different spectra of hosts. Further studies are needed in order to understand if these two lineages are associated with different disease outcomes.

Diversity of accessory genome of human and livestock-associated ST398 methicillin resistant Staphylococcus aureus strains

BackgroundMolecular typing of methicillin-resistant Staphylococcus aureus (MRSA) has documented the diversity of the genetic background of strains associated with healthcare (HA-MRSA), community (CA-MRSA) and livestock (LA-MRSA). The accessory and core-variable genomes of those strains however remain largely unknown. ObjectiveTo compare the genetic background and accessory and core variable gene content of ST398 LA-MRSA strains with those of HA-and CA-MRSA strains from the same region. MethodsRepresentative strains of HA- (n=21), CA- (n=13) and ST398 LA-MRSA (n=18) were selected from Belgian National Reference Laboratory collections. The accessory and core-variable genomes of these strains were characterized by a DNA-microarray composed of oligonucleotide probes targeting ∼400 resistance, adhesion and virulence associated genes. ResultsST398 strains displayed very homogenous hybridization profiles irrespective of their host origin. This ST398 genomic profile was moderately related to that of certain human HA- or CA-lineages but distinctively lacked several virulence- and colonization-associated genes implicated in carriage in humans, such as proteases and adhesins. No enterotoxin gene was found among ST398 strains. Differences were observed in the mobile resistance gene content of ST398 strains, including antibiotic resistance determinants. ConclusionLA-MRSA strains represent a homogenous lineage distinct from co-local HA- and CA-MRSA strains, especially in its accessory genome content characterized by a lack of human-associated virulence and adhesion determinants. The absence of detectable enterotoxin gene among ST398 LA-MRSA strains from a wide host range is reassuring regarding their foodborne pathogenic potential.

THE MOLECULAR PHYLOGENETIC AND GEOMETRIC MORPHOMETRIC EVALUATION OF MICRASTERIAS CRUX-MELITENSIS/M. RADIANS SPECIES COMPLEX1

We investigated nine strains of the Micrasterias crux-melitensis (Ehrenb.) Hassall ex Ralfs and M. radians W. B. Turner species complex. A combination of molecular, morphological, and geometric morphometric data was used to reveal the patterns of their phenotypic and phylogenetic differentiation. The molecular data based on internal transcribed spacer (ITS) rDNA, glycine transfer RNA (trnGuuc) intron, and SSU rDNA sequences revealed three phylogenetic lineages. One of them comprised the six European and North American strains that were morphologically identified as M. crux-melitensis. Phenotypic data illustrated high morphological variability of strains within this genetically homogenous lineage that spanned several traditional infraspecific taxa, including strains corresponding to M. crux-melitensis var. janeira (Racib.) Gronblad and M. crux-melitensis var. superflua W. B. Turner, whose morphometric characteristics profoundly differed. Three strains of M. radians formed two separate phylogenetic lineages corresponding to traditional varieties M. radians var. evoluta (W. B. Turner) Willi Krieg. and M. radians var. bogoriensis (C. J. Bernard) G. S. West. The morphological types corresponding to the former variety have, so far, only been reported from Africa. Therefore, we cannot preclude that geographic isolation may play a role in species differentiation of relatively large freshwater protists, such as Micrasterias.

Allozymes and mtDNA Reveal Two Divergent Lineages in Orchestia cavimana (Amphipoda: Talitridae)

Abstract We report on allozyme and mitochondrial DNA variation in 13 populations of the supralittoral amphipod Orchestia cavimana. The species colonizes fresh and brackish shores of Northern Africa, Near East and throughout Europe. Populations were sampled across a large part of the species range (Cyprus, Turkey, northern and southern Italy, United Kingdom and Germany), including the type locality (Cyprus). Both sets of molecular markers agree in identifying two divergent lineages. A first lineage includes populations from Cyprus and Turkey. A second genetically homogeneous lineage groups Italian, English and German populations. Genetic divergence between the two lineages is high (DNei = 0.720 ± 0.06 for allozymes and K2P = 0.191 ± 0.001 for mitochondrial DNA) and comparable to that among morphologically distinct congeneric species reported for the same set of molecular markers.

Effects of Reconstituted Collagen Matrix on Fates of Mouse Embryonic Stem Cells Before and After Induction for Chondrogenic Differentiation

Embryonic stem (ES) cells are pluripotent cells with great potential in regenerative medicine. However, controlling their differentiation toward homogeneous lineages is challenging. In this study, we aim to investigate the effects of reconstituted 3D collagen matrix on the fates of mouse ES (mES) cells before and after induction for chondrogenic differentiation. Specifically, mES cells were encapsulated and cultured in 3D collagen microspheres and exposed to induction signals at different time points. Growth characteristics and differentiation status of mES cells were then evaluated. Collagen microspheres provided a suitable microenvironment supporting mES cell growth and maintaining their undifferentiated status for certain period of time. At later time points, the proportion of undifferentiated mES cells gradually decreased, accompanied by increasing proportions of mesenchymal progenitor cells. This suggests the inductive role of collagen matrix in differentiating mES cells toward mesenchymal lineages. Moreover, a lower initial collagen monomer concentration facilitated the differentiation of mES cells into chondrogenic lineages, while induction at a later time point associated with a more advanced stage of chondrogenic differentiation. This indicates that both the initial collagen concentration and the time to induce differentiation significantly affected the fates of mES cells. This study contributes to future development of ES cell-based therapies.

Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile

A multilocus sequence analysis of ten virulence-associated genes was performed to study the genetic relationships between 29 Clostridium difficile isolates of various origins, hosts and clinical presentations, and selected from the main lineages previously defined by multilocus sequence typing (MLST) of housekeeping genes. Colonization-factor-encoding genes (cwp66, cwp84, fbp68, fliC, fliD, groEL and slpA), toxin A and B genes (tcdA and tcdB), and the toxin A and B positive regulator gene (tcdD) were investigated. Binary toxin genes (cdtA and cdtB) were also detected, and internal fragments were sequenced for positive isolates. Virulence-associated genes exhibited a moderate polymorphism, comparable to the polymorphism of housekeeping genes, whereas cwp66 and slpA genes appeared highly polymorphic. Isolates recovered from human pseudomembranous colitis cases did not define a specific lineage. The presence of binary toxin genes, detected in five of the 29 isolates (17 %), was also not linked to clinical presentation. Conversely, toxigenic A-B+ isolates defined a very homogeneous lineage, which is distantly related to other isolates. By clustering analysis, animal isolates were intermixed with human isolates. Multilocus sequence analysis of virulence-associated genes is consistent with a clonal population structure for C. difficile and with the lack of host specificity. The data suggest a co-evolution of several of the virulence-associated genes studied (including toxins A and B and the binary toxin genes) with housekeeping genes, reflecting the genetic background of C. difficile, whereas flagellin, cwp66 and slpA genes may undergo recombination events and/or environmental selective pressure.

The generation of MHC class II gene polymorphism in the genus Mus

Nucleotide sequences of exon 2 from 46 alleles of the murine MHC class II Aβ gene derived from 12 rodent species were determined using the polymerase chain reaction (PCR). This exon (273 bp) exhibited extensive nucleotide sequence diversity that did not follow predicted phylogenetic relationships. The nucleotide sequence diversity of exon 2 was focussed predominantly in five discrete sub-domains which contained polymorphic sequence motifs. These sub-domains each encode a segment of the antigen binding site of MHC class II molecule. The alleles in each sub-domain were organized into homogeneous lineages by parsimony analysis. The alleles in these lineages were very stable over long evolutionary periods. Some alleles derived from mice and rats have the same sequence motifs, indicating that they have been retained as polymorphisms since prior to the separation of the genus Mus and Rattus (10 Myr). Our findings indicate that polymorphism of MHC class II genes are generated by a combination of two processes. The steady accumulation of point mutations has produced highly divergent polymorphic sequence motifs in each sub-domain. This diversity is extensively amplified by repeated intra-exonic recombinations that shuffle these polymorphic sequence motifs into many, novel, allelic combinations. These intra-exonic recombinational events do not seem to occur at a extraordinary rate, or at specific sites within the exon. The protein sequences encoded by the polymorphic sequence motifs differed on average in 39.3% of amino acids and 67.3% of these substitutions were non-conservative. These findings indicate that they encode protein segments in the antigen binding site with distinct chemical properties. Codons for the antigen binding site have accumulated more non-synonymous (amino acid replacement) substitutions than synonymous (silent) substitutions, whereas the opposite is true for the non-antigen binding site codons in exon 2. All these results suggest that diversity is selectively maintained within the antigen binding sites. We propose that a special mechanism (termed divergent allele advantage), together with overdominant selection, accounts for the maintenance of a large number of highly divergent alleles in natural mouse populations. The extraordinary conservation of sequence motifs suggest that purifying selection may operate on MHC genes to selectively maintain certain sequence motifs and possibly combinations of specific motifs.