Repetitive DNA sequences as an insight into Aeglidae (Crustacea, Anomura) evolution

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M. E. D’Amato and D. Corach

Maria Eugenia D’Amato Daniel Conch Servicio de Huellas Digitales Geneticas and Ciitedra de GenBtica y Biologia Molecular, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Argentina

Elecrrophoresis 1997, 18, 1666-1611

Repetitive DNA sequences as an insight into Aeglidae (Crustacea, Anomura) evolution The evolutionary relationships of five Atlantic Aeglidae species (Aegla neuquensis affinis, A . humahuaca, A . jujuyana, A . platensis, and A . uruguayana) were studied by (i) satellite DNA analysis using a restriction enzyme digestion and hybridization pattern approach and (ii) genome screening by using randomly amplified polymorphic DNA (RAPD) typing. The identical restriction patterns and intense interspecific hybridization patterns obtained in this study strongly suggest a recent cladogenetic event for the Aeglidae. The speciesspecific amplification products which were detected using RAPD markers allowed species characterization. A total of 49 amplification products were used to construct trees by cluster analysis. The new scheme agrees in part with previous proposals based on biogeography and morphology. We considered that the subdivision northwestern-platensis species was probably due to the rising of the Andes, which started in the Middle Miocene. Divergence due to altitude is suggested by the different altitudinal distribution of three northwestern species along the same river. The possible role of selection by ecological factor/s was observed at the population level in A.Jujuyana, which has a wider altitudinal range distribution. RAPD markers revealed a high level of intraspecific diversity and important genetic flow among populations. However, a few markers showed significant differences in frequency or H between the lowermost population and the other populations, located in a different biogeographical region. The differences were not in relation to geographical distance, and we interpreted them as being due to selection. Repetitive sequences constitute an important reservoir of genetic variation, and these results show their usefulness in testing and proposing evolutionary hypothesis in crabs. These sequences seem to have played an important role in aeglid evolution. Ecological factors related to altitude have probably influenced macro- and microevolutionary processes, at least in northwestern species.

1 Introduction The Aeglidae (Decapoda, Anomura, Galatheoidea) is a monotypic family endemic to South America which contains the unique fresh water anomurans. The group is composed of approximately 40 species [l] which inhabit temperate and subtropical waters in the Pacific slope (Chile) and Atlantic slope (Argentina, Bolivia, Brazil, Paraguay, and Uruguay). Controversial aspects of their evolution and systematics were previously addressed using morphological or biogeographical data. The origins of the Aeglidae was traced to Late Cretaceous, from a marine New Zealand fossil record [2], and dispersal into lower latitudes probably took place in early Oligocene [3]. The systematic position of the Aeglidae (superfamily Galatheoidea) within Anomura remains controversial with arguments made for close evolutionary links to hermit crabs (Paguroidea and Coenobitoidea) [l] and to the primitive paguroids [4]. At the genus level, information on evolutionary relationships among species is sparse. For the Argentinian species (Atlantic species), four major morphological groups can be recognized which correspond to specific geographic distributions Correspondence: Dr. Maria Eugenia D’Amato, Servicio de Huellas Digitales Geneticas, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956-1 13 BA, Argentina (Fax: +54-1-964-8282)

Nonstandard abbreviations: RAPD, randomly amplified polymorphic DNA; UPGMA, unweighted pair group method with arithmetic averages Keywords: Aeglidae / Evolution / Repetitive DNA sequences / Randomly amplified polymorphic DNA

0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1997

[5--81. At the species level, morphological variability is high and this often hinders identification [9-131.

This study of the evolutionary relationships of the Atlantic species Aegla humahuaca and A . jujuyana (the Northwest Group), and A . neuquensis aflinis, A . platensi:r, and A . uruguayana (the platensis group) is based on: (i) satellite DNA analysis using restriction enzyme digestion and hybridization patterns, and (ii) genomr: screening by randomly amplified polymorphic DNA (RAPD) typing. Our results on highly repetitive DNA sequences did not provide useful information on the systematic position of the Aeglidae within Anomura. Nevertheless, all aeglids that were examined showed almost identical patterns and intense inter-specific hybridization, suggesting a recent cladogenetic event [14]. Prelirninary observations on aeglid cladogenesis using genome screening with RAPD markers and cluster analysis largely agreed with the morphological schemes [7, 81. As an insight into microevolutionary processes, we examined the population genetics of one northwestern species, Aegla jujuyana, using RAPD. The geographic distribution of this species is restricted to the Rio Grande basin in northwestern Argentina, where it is found in two different biogeographical provinces: (i) the prepuna (semiidesert between 1500 and 3000 m altitude), and (ii) the yungas (foggy forests) and its valleys (- 1200 m altitude). Although no subdivision among populations wa‘s found [15], significant differences in the frequency of some genetic markers between the yungas and prepuna populations suggest that selection due to altitude or related ecological factor/s [16] may have occurred. 0173-0835/97/090Y-1666 $17.50+.50/0

Elecrrophoresis 1997, 18, 1666-1671

Repetitive DNA sequences in Aeglidae evolution

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bitoidea, Diogenidae) were used as samples. All sampling sites were located in Argentina as follows: Humahuaca (23" 12' S, 65" 21' W, 2940 m) for A . humahuaca; 2.1 Animals from north to south along 120 km of the Rio Grande, Aeda hmahuaca and A . iWuYana (the northwestern Jujuy province, northwestern Argentina at Humahuaca, group), and A . n e w e n s i s affinis, A . piatensis and A . uru- Posta de Hornillos (-2300 m), Purmamarca (- 2100 m) guayana (the platensis group), Pachycheles haigae (Gala- and Rio Chico (24" 11' S, 65" 18' W, 1200 m) for A . theoidea, Porcellanidae), Pagurus gaudichaudi (Pagu- jujuyana; Yavi (22" 0 8 S, 65" 28' W) for A.n.a#inis; Rio roidea, Paguridae) and Loxopagurus loxochoelis (Coeno- Loro (26" 42' S, 65" 9' W) for A . platensis; and Arroyo el Pescado (33" 59' S, 57" 46' W) for A. uruguayana (Fig. 2 ) . For the RAPD and dot blot analysis, A. jujuyana individuals were sampled from the Rio Chico population. The marine species Pachycheles haigae (Galatheoidea, Porcellanidae), Pagurus gaudichaudi (Paguroidea, Paguridae) and Loxopagurus loxochoelis (Coenobitoidea, Diogenidae) were collected in Mar del Plata (38" 00' S, 57" 32' W).

2 Materials and methods

2.2 DNA extraction DNA was extracted from gonads as described previously [17]. Southern blots: DNA (4 mg) was digested with BamHI, EcoRI, HaeIII, HindIII, and TaqI Gibco BRL (Bethesda Research Laboratories, Maryland) and gel electrophoresis [141 and prehybridization [ 181 were performed

Figure 1. Hybridization patterns obtained with the probe A. jujuyana 400 bp HaelIl fragment. DNA of (1) A.n.affinis, (2) A. jujuyana, (3) A . platensis and (4) A. uruguayana was digested with HaeIII (A) and Hin-

dIII (B).

36"

2

40'

Figure 2. Sampling sites for A.n.affinis (1, Yavi), A. humahuaca (2, Humahuaca), A . jujuyana (3, Rio chico), A. platensis (4, Rio Loro) and A. uruguayana (5, Arroyo el Pescado). Box frames Rio Grande along distribution area of A. jujuyana.

Figure 3. Amplification products of A. humahuaca (lanes 1-3), A. jujuyana (4-6). A.n.affinis (7-9), A. platensis (10-12) and A . urnguayana (13-15) obtained with primers (A) OPA-02, (B) OPA-07, (C)

OPA-11 and (D) OPA-14.

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Table 1. Amplification products obtained for 3 individuals per species with the 10-mer primers OPA-02, OPA-07, OPA-11 and OPA14, Species-specific markers are shown in bold; bp, base pairs.

OPA 02-2 OPA 02-3 OPA 02-1 OPA 02-5 OPA 0 2 4 OPA 02-7 OPA 02-8 OPA 02.9 OPA 02.10 OPA 02-1 1 OPA 02-12 OPA 02-13 OPA 02-14 OPA 02-1 5 OPA07-1 OPA07.2 OPA 07-3 OPA 074 OPA 07-5 OPA 0 7 6 OPA 07-7 OPA 07-8 OPA 07-9 OPA 07-10 OPA 07-11 OPAO7.12 OPA 07-1 3 OPA 07-14 OPA11-1 OPA 11-2 OPA 11-3 OPAll4 OPA 11-5 OPA 1 1 4 OPA 11-7 OPAll-8 OPA 11-9 OPA 14.1 OPA 14-2 OPAAlcI OPA 1 4 4 OPA 1 4 5 OPA 14.6 OPA 14-7 OPA 1 4 4 OPA 1 4 4 OPA 14-10 OPA 14-1 1 Total

Plectrophoresis 1997, 18. 1666-1671

M. E. D'Amato and D. Corach

24W

2220 2030

1870 1730 1630 1500

13m 1240 1150 1080 920 800

740 2480 2050 1970 19W

17m 1420 1100 960

eeo 810 720 660

440 400 2800 2230 1900 1700 1130 940 700 560 360 1680 1480 12.20 1020 980 940 MO 760 680

Table 2. Amplification products obtained with primers OPA 02 (OPA 02-1/7), OPA 04 (OPA 04-1/8), and OPA 09 (OPA 09-117) for A. jujuyana populations"'

Bands

bD

OPA 02-1 OPA 02-2 OPA 02-3 OPA 02 4 OPA 02-5 OPA 02-6 OPA 02-7 OPA 04-1 OPA 04-2 OPA 04-3 OPA 04-4 OPA 04-5 OPA 04-6 OPA 04-7 OPA 04-8 OPA 09-1 OPA 09-2 OPA 09-3 OPA 09-4 OPA 09-5 OPA 09-6 OPA 09-7

2100 1700 1400 1280 1200 1050 780 2350 1570 1400 1100 900 800 700 620 2300 2100 1900 1720 1500 1320 400

N=19 f RC 0.579 0.263 0.895 0.842 0.421 1 1 0.368 0.316 0.684 0.316 1

0.789 0.737 0.474 1 0.579 1 1

0

N=O fPU

N=lO fHO

0.444 0.222 1 0.667 0.556 1 1 0.556 0.222 0.778 0.556 1 0.889 0.889 0.667 1 0 1 0.667 0 1 1

0.6

N=9 f HU 0.778 0.333 0.444 0.778 0.444

0.3

1 0.9 0.2 1 1 0.4 0 0.3 0.3 1 0.8 0.9 0.6 1 0 1 0.2 0.2 1 1

1 1

0.333 0 0.778 0.667 1 0.889 1 0.667 1 0 1 0.333 0 1 1

1 1 a) f RC, f PU, fH0, and f HU a r e the band frequencies in Rio Chico. Purmamarca, Posta de Hornillos and Humahuaca populations, respectively; N, sample size.

450 350

1

1

using standard procedures. Hybridization was allowed to proceed overnight at 65°C. Probes used in this study were selected from the restriction patterns, isolated from gels [19] and 200 ng of each was labeled by a random priming reaction. After hybridization, membranes were washed twice at room temperature and twice at 65°C in 2 X saline-codium citrate buffer (SSC), and autoradiographed at -70°C in cassettes containing intensifying screens. 2.3 RAPD

PCR was performed in 30 mL volumes, with 1 U Tuq polymerase BRL, 5 pm primer (see below), 25 ng DNA, 2.3 mM MgCl,. Cycling: 94°C for 3 min, 35 cycles of 94"C, 1 min; 35°C for 1 min, 72°C for 2 min, and extension time of 10 min at 72°C. PCR reaction products were run in 1.5% agarose gels in 1 X Tris-borate-EDTA (TBE) buffer with ethidium bromide at 2 Vlcm. 2.4 Primers

10-mer oligos OPA-02 (5' TGCCGAGCTG 3'), OPA-04 (5' AATCGGGCTG 3'), OPA-07 (GAAACGGGTG), and OPA-09 (5' GGGTAACGCC 3'), OPA-11 (CAATCGCCGT) and OPA-14 (TCTGTGCTGG) (Kit A, Operon Technologies, Inc., Alameda, CA, USA).

kGi

D distance among five Aegla species. A binary character matrix was constructed from the presence absence of bands. A derived distance matrix was obtained by pairwise comparisons among all 15 individuals. Distance was calculated as D = 1-S [ZO]. Morphological species groups shown o n the right.

Figure 4. UPGMA cluster analysis of

2.5 Data analysis 2.5.1 Aeglidae systematics with RAPD A binary character matrix was constructed from the presencelabsence of bands (Table 2). A derived distance matrix was obtained by pairwise comparisons among all 15 individuals with Nei and Li, 1985 D distance [20], for further cluster analysis with unweighted pair group method with arithmetic averages (UPGMA) using NTSYS-pc 1.8 [21]. 2.5.2 Population analysis Population genetic parameters were calculated as in [22], and FST significance was tested by (x2) = 2 N FST (k-I) with (k-1) (s-1) degrees of freedom, where N is the total number of individuals sampled, k is the number of alleles, and s the number of subpopulations. Significant differences at a 5 O/o significance level between popula-

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Repetitive DNA sequences in Aeglidae evolution

ElrctrophorPsis 1997. 18, 1666-1671

Figure 5. Amplification products of 5 individuals from Rio Chico population with primers (A) OPA-02, (B) OPA-04, (C) OPA-09.

Table 3. Below diagonal: Rogers’ distances among Humahuaca (HU). Posta de Hornillos (HO), Purmamarca (PU), and Rio Chico (RC): above diagonal: distances in km between Dopulations HU

HO

PU

RC

HU

0

so

HO PU RC

0.044

64 14

124 74 60 0

0.039 0.0554

0 0.043 0.0552

0 0.039

tions were tested with two-sample t-tests between H and with a (x’) statistic between marker frequencies. Marker frequencies were used to calculate Rogers’ distance [23] between populations. Distance matrix was analyzed with NTSYS-pc 1.8 for UPGMA tree construction.

3 Results 3.1 Aeglidae systematics 3.1.1 Systematic position within Anomura

The systematic position of the Aeglidae was examined by comparing its restriction enzyme digest and hybridization patterns of highly repetitive DNA sequences [14] with those of other Anomura, Brachyura and Caridea. The most outstanding results were the high similarity within Aeglidae and the apomorphic characteristic of their highly repetitive DNA. The family was characterized by a monomeric repeating unit of 400 bp with restriction sites for HaeIII, HindIII (Fig. l), and EcoRI, and sites for TaqI, each aproximately 200 bp. All species showed identical patterns, with the exception of A . jujuyana and A . uruguayana / HindIII, where most intense bands correspond not to the monomer but to the dimeric or the trimeric repetitions. 3.1.2 Systematics within Aegla genus

The RAPD technique was used to more clearly define the genetic relationships within the genus and species characterization and tree construction were possible using RAPD markers. PCR genomic DNA amplification of three individuals/species with four different primers (OPA-02, OPA-07, OPA-11 and OPA-14) resulted in 49 different amplification products, 15 of which were species-specific (Fig. 4 and Table 2). Distance matrix with D = 1-S (S of Nei and Li, 1985) was used to obtain the UPGMA tree (Fig. 5). The UPGMA tree showed a high goodness of fit (cophenetic correlation coefficient r = 0.97) and largely agrees with previous morphological or biogeographical data [7, 81. However A.n.affinis, which inhabits northwestern Argentina, clusters with northwest

0 051

,

Roprr’gmchc &IJIUC

0 wz

-

0 045

0 042

0 037

1

PlCIVJlld

Yungas’ valley

Figure 6. UPGMA analysis of Rogers’ genetic distances between A. jujuyana populations. HU, Humahuaca; PU, Purmamarca; HO, Posta de Hornillos; RC, Rio Chico; Prepuna and Yungas are the biogeographical provinces to which the populations belong.

species. This is in agreement with strict biogeographical data. 3.2 Population analysis

Populations of A . jujuyana were sampled at various sites along a 120 km section of the Rio Grande in northwestern Argentina (see Section 2 and Table 3). From a total of 22 different amplification products (Fig. 5) obtained with primers OPA-02, OPA-04 and OPA-09, seven were constant over all populations (Table 2). Population genetic parameters were calculated as in [22]. The analysis was restricted to bands with a frequency lower than 1-3/N, where N is the sample size). Consequently, the analysis of population subdivisions had to be restricted to bands OPA 02-2, 02-5, 04-1, 04-2, 04-4, 04-8, 09-2, and 09-5. The Fst estimate of 0.0125 was not significant (x’ = 9.4, df = 24, P > 0.05), indicating that an among-population differentiation was not detected. However, some of the bands excluded from this analysis were found at frequencies which were low enough to calculate a reliable H in other populations (e.g., bands OPA 02-1, 04-3, 09-4), and some of these showed different frequencies throughout populations. In order to test the significance of these differences (5% level of significance) we performed two-sample t-tests with all H values, and x2 statistic with the high frequency bands (OPA 02-3 and 09-4). Bands 02-3, 04-2, 09-2, and 09-4 showed differences between some populations, in which 9 out of 11 differences occurred in the Rio Chico population [15]. UPGMA clustering of genetic Rogers’ distances further illustrated population relationships (Fig. 7). Although distance values (Table 4) between populations were low, prepuna populations appear more closely related. The same result was obtained with D = 1-Svalues ( S of [20]).

4 Discussion Satellite DNAs are known as fast evolving components of the genome which are not subjected to selective pressure. As such, they can provide qualitative evidence of evolutionary interest since closely relatcd species often

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M. E. D’Amato and D. Corach

Electrophoresis 1997, 18, 1666-1671

show homologies in their profiles. Crustacean genomes at -500 m). This observation suggests a clinal diverare rich in satellite DNA sequences, and homology and gence due to altitude or related ecological factor/s, assoconservation of these sequences were reported for Deca- ciated thus to the rising of the Andes that started in the poda [24, 251. We previously detected low levels of Middle Miocene. hybridization between Pagurus and Loxopagurus hermit crab satellites, which failed to hybridize to Aeglidae and From upper Oligocene to Middle Miocene (- 16.3-10.4 Porcellanidae (Galatheoidea) DNA [14]. This may be due million years), a large portion of South America was to conservation of repeated sequences from before diver- covered by a continental sea with - probably - low salt gence of right-handedileft-handed hermit crabs. concentration [33, 341. Although aeglids might have disAeglidae satellite DNA was not shared by any other Ano- persed into lower latitudes in early Oligocene [3], diversimura species and can be considered an apomorphy fication probably took place later [16], in relation to the which is characteristic of the family [14]. Satellite DNA Andes rising, at least for northwestern species. A . was not informative for the systematic position of this jujuyana is the northwestern species that displays the group within Anomura. The satellite DNA profiles of all widest distribution in northwestern Argentina - in terms aeglid species were similar and no species-specific profile of ecological factors to which they are subjected -, and was identified. The patterns of periodicity in A. jujuyana the largest altitudinal range. and A . uruguayana, which had a higher intensity of dimers and trimers, may result from a rolling circle The most outstanding observations on these populations amplification process (for more detail see [26]). This are: (i) the important gene flow that connect them, probevent could have taken place in northwest and platensis ably due to dispersion during the rainy season [15], and groups independently. The similarity in satellite DNA (ii) the differences observed between the valley and the profiles and the high levels of inter-specific homology, prepuna populations, in few markers frequency and using the 400 bpiHaeIII repeating unit as a probe, sug- genetic Rogers’ distance cluster analysis. We interpreted gests a close evolutionary relationship among aeglids these differences as selection affecting some markers [15, 161 rather than random variation. Repetitive sequences ~41. seem to have played an important role in Aeglidae evoIn view of their potential preference for repetitive DNA lution. Its satellite seems to be an apomorphy, which is amplification, RAPD markers were used for further anal- conserved among species, and strongly suggests that ysis. Although it was proposed that the technique scans Aeglidae cladogenesis is relatively recent. Analyzed spethe genome randomly [22], RAPDs may in fact tend to cies are closely related and ecological factors related to originate from repetitive DNA [27]. The structure of altitude probably influenced macro- and microevoluRAPD products is suggestive of transposons - the inter- tionary processes, at least in northwestern Argentina. vening sequences between inverted repeats. Other repetitive sequences with inverted repeats could also be tar- We specially thank Dr. H. Dopazo and Dr. A . L. Cione for gets for RAPD primers. In Crustacea, two examples illu- helpfull commentaries on data analysis and critically strate this: (i) RAPD markers that show differential dis- reading of the manuscript. tribution among Pennaeus populations contained microsatellites (as well as an open reading frame) [28] and (ii) Received February 26, 1997 Daphnia RAPDs which were used for microsatellite isolation [29]. The use of RAPDs in phylogenetic analysis highlights some theoretical problems such as the homol- 5 References ogy of the character “band present”, asymmetrical proba[ l ] Martin, J. W., Abele, L. G., Smithsonian Contribution to Zoology bility of gains and losses of bands, band absences may 453, Smithonian Institution Press, Washington, DC, 1988, represent different character states (e.g. inversion, delepp. 1-46. [2] Feldmann, R. M., N. Z . J. Geo. Geophy. 1984, 27, 379-385. tion, point mutations, etc.) [30-321. For this reason, re[3] Feldmann, R. M., in: Gore, R. H., Heck, K. L. (Eds.), Crustacean striction of the technique to organisms that are closely Biogeography, Balkema Publishers, Rotterdam 1986. related is recommended, and not to infer systematic rela[4] Bouvier, M. L., Faune de France 1940, 37, 1-399. tionships at higher taxonomic ranks [30,32], which seem [ 5 ] Lopretto, E. C., Neofropica 1978, 24, 55-68. to be the Aeglidae case. For the same reason, previous [6] Lopretto, E. C., Neotropica 1979, 24, 9-22. empirical phylogenetic hypothesis should be considered. [7] Lopretto, E. C., Physis (Buenos Aires) 1980, B39, 37-56. In our case, the clusters ( A . humahuacad. jujuyana) and (A. platensisd. uruguayana) obtained with RAPD markers are largely consistent with biogeographical and morphological data, and only the position A.n.a@nis is in disagreement with previous proposals [7, 321. However, with this preliminary analysis we cannot assign a certain group to this species, and a more detailed analysis with other genetic sources of information should be performed. The Northwest group is composed of four species, A . franca, A . humahuaca, A . jujuyana, and A . sanlorenzo, and the three latter are distributed altitudinally along the same basin. ( A . humahuaca from 30002100 m, A . jujuyana from 1000 to 3000 m, A . sanlorenzo

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