Large Amounts of Genetic Divergence among Italian Species of the

Molecular Phylogenetics and EvolutionVol. 17, No. 3, December, pp. 456–461, 2000doi:10.1006/mpev.2000.0854, available online at http://www.idealibrary.com on

Genus Orchesella (Insecta, Collembola) and the Relationshipsof Two New Species

Francesco Frati, Emiliano Dell’Ampio, Stefania Casasanta,Antonio Carapelli, and Pietro Paolo Fanciulli

Department of Evolutionary Biology, University of Siena, via P. A. Mattioli 4, 53100 Siena, Italy

Received February 25, 2000; revised August 11, 2000; published online November 30, 2000

al., 1994) have helped to confirm the diagnosis of spe-

The phylogenetic position of two putative new spe-

cies of the collembolan genus Orchesella was investi-gated by comparison with four other Italian species ofthe genus using a fragment of the mitochondrial geneencoding for subunit I of cytochrome c oxidase (COI).The gene showed the well-known A 1 T bias, typical ofinsect mitochondrial DNA, although A 1 T content wasnot as high as that observed in species belonging tomore derived insect orders. The large number of vari-able sites in 3rd codon positions (85.2% variable) sug-gested that these sites contain significant homoplasydue to multiple hits. Despite the lack of morphologicaldifferentiation, the COI portion examined shows re-markable levels of genetic divergence between the pu-tative species and their closest relatives. Phylogeneticanalysis suggests that one of the putative new speciesis related to O. villosa, whereas the other is includedin a clade with O. cincta and O. ranzii. The species O.chiantica appears to be related to O. villosa, agreeingwith previous allozyme data. © 2000 Academic Press

Key Words: phylogeny; likelihood; parsimony; COI;molecular evolution; gene trees.

INTRODUCTION

Species of the holoarctic genus Orchesella (Entomo-bryidae) inhabit the uppermost layer of the forest litterwhere they constitute a key factor in the recycling oforganic matter. Despite the fact that Orchesella speciesare among the largest collembolans, morphologicalcharacters of taxonomic importance are reduced tomainly the arrangement of sensorial hairs (chaetotaxy)and to pigmentation patterns (Stach, 1960; Yoshii,1989), which, however, are useful only for species di-agnosis and not for reconstructing phylogenetic rela-tionships. This is due to the high levels of intraspecificvariability which affect both chaetotaxy and pigmenta-tion and are sometimes correlated with their life cycle(Parisi, 1960; Szeptycki, 1979). Allozyme data (Frati et

1055-7903/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

456

cies based on pigmentation patterns and suggestedthat additional undescribed species may be present insome Italian localities. It was also shown that Plio–Pleistocene geographical rearrangements which oc-curred in the Italian Peninsula may have generatedlarge levels of differentiation between geographicallyisolated populations, possibly providing the basis formicrospeciation processes. Similarly, DNA sequencedata from the mitochondrial gene cytochrome c oxidaseII (COII) were used to produce a phylogenetic hypoth-esis which was partially in agreement with allozymedata (Frati et al., 1997). In this paper, sequence anal-ysis of a portion of the mitochondrial gene encoding forsubunit I of cytochrome c oxidase (COI) provides evi-dence for the relationships of two putative new speciesin the genus Orchesella with four of the most commonItalian species of this genus.

INTRA- AND INTERSPECIFIC VARIABILITYIN ORCHESELLA

Specimens were collected in several sites along theItalian Peninsula and six species were found: Or-chesella villosa, O. cincta, O. ranzii, O. chiantica, andtwo putative new species, Orchesella sp.-ELB and Or-chesella sp.-MTB (ELB: Elba Is., off the Italian westcoast; MTB: Mt. Bondone, northeastern Alps). Six geo-graphically isolated populations from central Italy(FEN, SIE, AMI, LIV, CGR) and an off-shore island(GIG) were sampled for O. villosa. Three populationswere considered for O. chiantica (AMI, BRO, APU), alllocated in a range of about 200 km (see Frati et al.,1994 for a map).

As expected for an insect mitochondrial gene, basecomposition is biased toward As and Ts (66.1%: Table1), with the bias being highest in 3rd codon positionsites (80.2%). A similar value (68.8%) is found in theonychiurid collembolan Tetrodontophora bielanensis,whereas the corresponding fragment of Drosophila

c4o

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TABLE 1

p

457LARGE GENETIC DIVERGENCE IN Orchesella

yakuba is more A 1 T-rich (70.7%). The 2nd and 3rdodon positions are also remarkably rich in Ts (over0% in almost all individuals), suggesting the presencef a strong codon usage bias.Whereas base composition across all sites is quite

omogeneous (Table 1), 3rd codon positions are muchore heterogeneous than 1st and 2nd positions, re-ecting their higher rate of variability. Overall vari-bility among the 13 sequences of Orchesella, in fact, is

35.8% (187 of 522 sites are variable; Table 1). Most ofthe variability is concentrated in 3rd codon positions,with 2nd codon positions being the least variable. Theamount of variability observed across Orchesella issomewhat higher than that found in other phylogeneticstudies involving the COI gene in congeneric species ofgrasshoppers (Lunt et al., 1998) and bumblebees (Ped-ersen, 1996), suggesting the possibility that the COIgene in Orchesella evolves at a higher rate than inother insect genera. A similar result was obtained froma set of COII sequences from five different species ofOrchesella (Frati et al., 1997) where the fraction ofvariable sites was very similar (34.4%). There are ex-ceptions to this, however, namely in the whitefly genusBemisia where over 55% of positions of a COI fragmentwere found to be variable (Frohlich et al., 1999).

Heterogeneity in base composition and overall rateof variability are crucial parameters to consider whenone wants to study the evolution of a gene, and both ofthem are very high in 3rd codon positions. A highcompositional heterogeneity suggests that these sitesmight be evolving under nonstationary conditions andthis should be taken into account when phylogeneticanalysis is performed with models of evolution whichassume stationarity of base composition across alltaxa. Similarly, the fact that over 85% of 3rd codonpositions are variable suggests that multiple substitu-tions are likely to occur in these sites, masking orconfusing the phylogenetic signal.

Nucleotide Composition and Distribution of VariaStudied Divided

All sitesa 1st codon p

A C G T A C

Mean 0.290 0.164 0.176 0.371 0.337 0.134 0Compositional biasb 0.214 0.171Test of homogeneityc x2 5 12.975, P 5 0.999 x2 5 2.839, PVariable sitesd 0.358 0.172 (16

a DNA extraction protocols were according to Simon et al. (1991)erformed with the primers COIf (59-CCTGCAGGAGGAGGAGATC

fragment (with no indel) was 522-bp long, corresponding to positionsand Wolstenholme, 1985). Sequences can be retrieved from GenBan

b Calculated according to Irwin et al. (1991).c As implemented in PAUP* (Swofford, 1998).d Values in parentheses are distribution of variable sites across ea

Intra- and interspecific degrees of divergence (uncor-rected) differ extensively among the species. Withinthe species O. villosa and O. chiantica, the uncorrectedpercentage of sequence divergence between popula-tions is lower than 6%, with the exception of compari-sons involving O. villosa-CGR (9–10%). Percentage se-quence divergence between species varies between 10and 21%, whereas the divergence between any Or-chesella sequence and T. bielanensis almost alwaysexceeds 20%. These values are much higher if geneticdistance is estimated, as it should be, according to aspecific model of evolution that corrects for multiplesubstitutions. The larger the genetic distance, thegreater the effect of the correction. A comparison withother studies on the COI gene shows that similar val-ues of interspecific genetic divergence (approximately20%) have been observed in genera of Coleoptera (Funket al., 1995; Juan et al., 1995, 1996; Langor and Sper-ling, 1995; Funk, 1999; Garin et al., 1999) and Hy-menoptera (Mardulyn and Whitfield, 1999), whereasmuch lower values have been found in genera of Lep-idoptera (Brower, 1994; Brown et al., 1994; Brunton,1998), Thysanoptera (Crespi et al., 1998), and Diptera(Frohlich et al., 1999; Ståhls and Nyblom, 2000). Theobserved heterogeneity of interspecific sequence diver-gence in different insect genera may be explained inseveral ways. On the one hand, the COI gene mayeffectively evolve at quite different rates in differentlineages, possibly as a consequence of changes in theselective pressures affecting the gene. This is concor-dant with the evidence that mitochondrial genes mayin fact have different rates of evolution in differentlineages (Crozier et al., 1989). On the other hand, theobserved differences in sequence divergences in conge-neric species is certainly biased by the different signif-icance of the genus rank in different orders or evenfamilies. The boundaries of a genus are usually set onthe presence of specific morphological differences and

e Sites Observed in the 13 Orchesella SpecimensCodon Position

tion 2nd codon position 3rd codon position

T A C G T A C G T

1 0.238 0.164 0.224 0.171 0.441 0.367 0.133 0.065 0.4350.255 0.403

1.000 x2 5 0.749, P 5 1.000 x2 5 41.800, P 5 0.233) 0.046 (4.3%) 0.852 (79.7%)

hahjahan et al. (1995), and Sunnucks and Hales (1996). PCR was39) and COIa (59-AGTATAAGCGTCTGGGTAGTC-39). The aligned12 to 2733 in the Drosophila yakuba mitochondrial sequence (Clarynder Accession Nos. AJ272251–63.

codon position.

blby

osi

G

.29

5.0%

, SC-22

k u

ch

the evolution of morphological traits has been demon-

458 FRATI ET AL.

strated to proceed at a different rate than the evolutionof DNA sequences in many instances (e.g., Wu and Li,1985; Britten, 1986; Sturmbauer and Meyer, 1992),resulting in the observed phenomenon of heteroge-neous interspecific divergence in different genera.

In collembolan classification, and especially amongOrchesellinae, species are often identified on the basisof pigmentation (Stach, 1960; Yoshii, 1989) and generaare defined by other anatomical characters. However,if species diagnosis is complicated by intraspecific vari-ability and/or presence of sibling species (as demon-strated by genetic data: Carapelli et al., 1995), theboundaries of genera are also somewhat unstable. As aconsequence of these difficulties, but possibly influ-enced by relatively frequent microspeciation events,there are large genera containing a high number ofspecies. Also, allozyme data in Orchesella have shownlarge amounts of interspecific genetic divergence (Fratiet al., 1994), whereas mtCOII sequence data revealedthat collembolan species and genera have levels ofdivergence much higher than those observed in otherinsect groups (Frati et al., 1997).

PHYLOGENETIC PATTERNS: THERELATIONSHIPS OF THE NEW SPECIES

Phylogenetic analysis on aligned nucleotide andamino acid sequences (Clustal W: Thompson et al.,1994) was performed under minimum-evolution (ME),maximum-parsimony (MP), and maximum-likelihood(ML). In all analyses, the sequence of the collembolanTetrodontophora bielanensis (GenBank Accession No.AF272824) was used as an outgroup. In ML searches,the model of evolution was selected according to theiterative search strategy proposed by Swofford et al.(1996) (see: Frati et al., 1997; Sullivan et al., 1997, forexamples). For nucleotide sequences, two data setswere considered: one containing the complete 522-bpsequence (Fig. 1) and one from which the highly vari-able and potentially saturated 3rd codon positionswere removed (Fig. 2).

The various gene trees resulting from the phyloge-netic analyses of the COI gene allow discussion ofspecific hypotheses on the placement of the new unde-scribed species in relation to the other taxa. If thesegene trees are assumed to match the species phylog-eny, the picture resulting from all analyses suggeststhe relationship of Orchesella sp.-ELB with O. villosa.This is in agreement with morphological and allozymedata (Frati et al., 1994). The failure to find support fora monophyletic clade including only O. villosa popula-tions may even suggest that the population from ELBdoes not belong to a different species, although (cor-rected) distance values with other populations are con-stantly above 0.200 and values among populations of

O. villosa never exceed 0.160. Species status, however,cannot be based on genetic distance. Given more data,the identification of additional genetic structure withinwhat is now O. villosa could justify breaking this taxoninto multiple species. In any case, a monophyletic cladeincluding only the population from ELB and all otherpopulations of O. villosa can be observed only when allsites are examined (Fig. 1), whereas when 3rd codonpositions are removed or amino acid sequences areconsidered the population from ELB belongs to an un-resolved clade including other species (Figs. 2 and 3).

On the other hand, there appears to be a strongersignal for the monophyly of O. chiantica, whose threepopulations always cluster together and share aunique aa change (Fig. 3). Its relationship with O.

FIG. 1. Maximum-likelihood reconstruction (PAUP*: Swofford,1998) using all nucleotide sites and based on the GTR 1 I 1 G model(Yang, 1994; Swofford et al., 1996; -Ln likelihood 5 2638.622; I 50.428; a 5 0.550); heuristic search with parameters estimated duringthe search. Branch lengths are maximum-likelihood estimates. Boot-strap values (100 replicates) above 50% are shown at the nodes: MLestimates (with parameters fixed as above) are boxed, MP estimates(with equal weight assigned to all sites) are above the node, and MEestimates (GTR 1 I 1 G-corrected distances with ML-estimatedparameters) are below the nodes.

ip(

taabacaat

1989), might be of lesser utility for phylogenetic recon-

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t

1mIsse(ep

459LARGE GENETIC DIVERGENCE IN Orchesella

villosa and Orchesella sp.-ELB, which is in agreementwith allozyme data (Frati et al., 1994), is not supportedn nucleotide-based analyses, but it is strongly sup-orted by three unique synapomorphic aa changesFig. 3).

With respect to the position of Orchesella sp.-MTB,here is strong evidence (including five synapomorphica changes) for its placement in a clade with O. cinctand O. ranzii (Figs. 1–3). All these species exhibit onelack and one white transversal bands across their IInd III abdominal tergites. This character, however,ould not be considered critical for their relationships,s it is also shown by the larger-sized species O. chi-ntica, suggesting that features of pigmentation pat-erns, while being useful for species diagnosis (Yoshii,

FIG. 2. Maximum-likelihood reconstruction (PAUP*: Swofford,998) using 1st and 2nd codon positions and based on the GTR 1 Iodel (Yang, 1994; Swofford et al., 1996; -Ln likelihood 5 885.780;5 0.791); heuristic search with parameters estimated during the

earch. Branch lengths are maximum-likelihood estimates. Boot-trap values (100 replicates) above 50% are shown at the nodes: MLstimates (with parameters fixed as above) are boxed, MP estimateswith equal weight assigned to all sites) are above the node, and MEstimates (GTR 1 I 1 G-corrected distances with ML-estimatedarameters) are below the nodes.

structions. Interestingly, a relationship between O.cincta and O. ranzii was detected also by using allo-zymes (Frati et al., 1994), but not by studying themitochondrial COII gene, with which O. ranzii wasshown as more closely related to O. villosa than to O.incta (Frati et al., 1997). In addition, a relationshipetween Orchesella sp.-BRO and O. ranzii is suggestedy three synapomorphic aa changes.Our analysis of the COI gene does not resolve rela-

ionships at the intraspecific level in O. villosa (Fig. 1);therefore, no hypothesis can be made on phylogeo-graphic relationships among populations of this spe-cies. A clearer pattern is observed in O. chianticawhere a well-supported relationship is established be-tween the populations from AMI and BRO (Fig. 1), tothe exclusion of APU. This latter population comes

FIG. 3. Parsimony analysis (with equal weight assigned to allsites) of amino acid sequences inferred from the Drosophila mito-chondrial code (de Brujin, 1983) (12 parsimony informative sites; 47steps; CI 5 0.957). Numbers at the nodes represent bootstrap sup-port values (100 replicates). Nodes with bootstrap values lower than50% were collapsed. Synapomorphic amino acid changes for signifi-cant clades are shown along the internodes with indication of thetype of change and the position in the sequence.

from the Apennine mountain range, whereas the re-

Omlwwscpuavcctcva

markers for the diagnosis of sibling species in the genus Isotomu-

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J

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L

M

P

460 FRATI ET AL.

maining two are from central and southern Tuscany.This analysis seems to suggest a geographic pattern inthe relationships among these populations, whose dif-ferentiation may reflect Plio–Pleistocene paleogeo-graphic rearrangements which appear to have influ-enced the levels of genetic differentiation on otherspecies of edaphic insects (Fanciulli et al., 2000).

CONCLUSIONS

Although the study of other European species ofrchesella could help to clarify the overall picture, thistCOI-based phylogeny allows description of some re-

ationships at the interspecific level. In particular, aell-supported monophyletic group can be recognized,hich includes O. ranzii, O. cincta, and Orchesella

p.-MTB. The other new species, Orchesella sp.-ELB,an be reasonably associated with O. villosa. Moreroblematic is the position of O. chiantica, whose pop-lations form a strong monophyletic clade, but syn-pomorphic aa changes support its placement with O.illosa and Orchesella sp.-ELB. The removal of 3rdodon position sites (which could be justified by theirompositional heterogeneity) leads to a loss of resolu-ion on the trees and to a change of the position of O.hiantica, which is placed as the sister taxon of O.illosa, in agreement with the reconstruction based onmino acid sequences.

ACKNOWLEDGMENTS

We are grateful to R. Dallai for helpful assistance. We also wish tothank C. Simon, in whose laboratory this project was started, foruseful comments on an earlier version of the manuscript. R. DeSalleand two anonymous reviewers also provided useful comments. Theresearch was supported by grants from the Italian M.U.R.S.T. (Pro-grammi di ricerca di rilevante interesse nazionale) and the Univer-sity of Siena (P.A.R. 1999) to F.F.

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