The Aconcagua River as another barrier to Liolaemus monticola ( Sauria : Iguanidae ) chromosomal races of central Chile

Univariate and multivariate statistical analyses of 28 meristic characters recorded for 19 samples of the lizard Liolaemus monticola monticola were used to compare geographical variation in morphology with chromosomal races north and south of the Maipo River, and north and south of the Aconcagua River in central Chile, plus some affluent. This extends a previous morphological study that confirmed that the Maipo River is a biogeographical barrier that separates chromosomal races “Southern, 2n=34” and “Northern, 2n=38-40”, and reports for the first time the importance of the Aconcagua River as another biogeographical barrier between the chromosomal races “Northern, 2n=38-40” and the “Multiple Fission, 2n=42-44”. The phenetic variation among samples is sufficient to differentiate the three chromosomal races, and also distinguishes populations within the chromosomal races such as the ones separated by the Colorado River, an affluent of the Aconcagua River. A possible historical sequence of events that accounts for the pattern of morphological differentiation is advanced.


INTRODUCTION
Rivers may be barriers to dispersal, the most effective ones being those that change their courses least often (Mayr 1963).Riverine barriers sometimes separate races (Lamborot & Eaton 1992, 1997) or closely related species (e.g., Haffer 1974); in other cases populations of the same race but conspicuously different in phenotype may be separated by a river.River size is generally a good predictor of differentiation (e.g., Pounds & Jackson 1981), and the differences across a river are greater at its mouth than in the headwaters as in the Patton's "riverine barrier hypothesis" The Aconcagua River as another barrier to Liolaemus monticola (Sauria: Iguanidae) chromosomal races of central Chile
Palabras clave: razas cromosómicas, variación morfológica, barreras geográficas, Liolaemus monticola, río Aconcagua.(Patton et al. 1994).An opposite situation was found for the Liolaemus monticola chromosome races north and south the Maipo River in which historical aspects as Pleistocenic glaciations indicates that this river acted as a barrier more strongly near the headwaters of the river than near its mouth (Lamborot & Eaton 1997).
However, it is difficult to know if the rivers are facilitating differentiation or simply limiting the re-expansion of a former macrogeographic disjunct.
Liolaemus monticola (von Müller & Helmich 1932) is a highly variable, endemic montane lizard species distributed along the temperate Andes, coastal and transversal ranges in Chile, between latitudes 30º and 40º S at altitudes between 900 and 2,300 m (Donoso- Barros 1966, Peters & Donoso-Barros 1970).This species displays a latitudinal gradient of karyotypic diversity (Lamborot 1993), and thus offers an ideal system for studying the possible evolutionary roles of various kinds of karyotypic modifications and modes of evolution in the differentiation of species.And because the dispersal of the L. monticola lizards is restricted by a number of rivers, it is possible to investigate the influence of several such barriers.Replicates of this sort are necessary for testing the hypothesis that differentiation is enhanced by intervening rivers.
The "Southern, 2n=34" and the "Northern, 2n=38-40" chromosomal races are clearly separated by riverine barriers; in the Coastal Range by the Maipo River, and in the Andes by the Maipo River and one of its affluent, the Yeso River (Lamborot 1991, Lamborot & Álvarez 1993).Hybridization between the races is found only in a narrow zone which was considered a zone of secondary contact, favored by man-made structures (Lamborot 1991).Multivariate analysis of meristic characters produced groups which coincided exactly with the separation of the chromosomal races, and thus confirmed the Maipo and the Yeso Rivers as river barriers (Lamborot & Eaton 1992, 1997).
C h r o m o s o m e a n a l y s i s s u g g e s t s t h a t t h e Aconcagua River has also been a barrier to this species, presently separating the "Northern, 2n=38-40" and the "Multiple Fission (MF), 2n=42-44" chromosome races (Lamborot 1998(Lamborot , 2001)).
Geological data for the region in Central Chile demonstrate that glaciation in the Pleistocene period was extensive (Brüggen 1950, Vuilleumier 1971, Caviedes 1972).The Aconcagua Valley (32º 50'S), located in the transition between the arid zone of the so-called "Norte Chico" and the temperate winter humid part of Middle Chile, has a history of glaciations similar to that of the Maipo River Valley.Thus if differentiation is enhanced by riverine barriers, we should expect the variability in the Aconcagua Valley to parallel that of the Maipo Valley.
In this contribution, we assess the degree of morphological divergence that has occurred within and between the two L. m. monticola chromosomal races "Northern, 2n=38-40" and "MF 2n = 42-44" (Lamborot 1998).We compare these populations with some representative localities belonging to the "Southern, 2n=34" and the "Northern, 2n=38-40" chromosomal races from the Andes and the Coastal Ranges.We test whether the derived "MF" differs in variability from the ancestral race, and the importance of the Aconcagua River as a barrier to the gene flow between races.Furthermore, we offer possible explanations for the present distribution of chromosomal races of L. m. monticola and the importance of the rivers as biogeographical barriers in the central Chile.
We also included samples of the "Southern, 2n=34" race localities (Lamborot & Eaton 1997) from the Coastal Range, Cerro Cantillana (14-CAN); from the Andes, Río Clarillo (18-CLAR), Volcán Sur (16-VSUR), Volcán Norte (17-VNOR), Yeso Sur (19-YSUR) and Los Queñes (15-QUE), which is 200 km south of the other populations.The Yeso Sur population is located 200 m from Yeso Norte, and is connected by a bridge which crosses the Yeso River; a few interracial hybrid individuals (Lamborot 1991, Lamborot & Eaton 1992) are included in the sample.Most individuals from each sample were karyotyped (Lamborot 1991(Lamborot , 1998 1) in the Andes, Coastal and Transversal ranges.Numbers enclosed in triangles represent sampling localities of the "Multiple Fission, 2n=42-44" race; those enclosed in circles represent sampling localities of the "Northern, 2n=38-40" race; those enclosed in double circles represent sampling localities of the "Northern modified 1, 2n=38-40" race, north and south the Colorado River, an affluent of the Aconcagua River; those enclosed in squares are localities of the "Southern, 2n=34" race; the circle and square together represents the zone of secondary contact.The sampling locality of Los Queñes (15) is not shown on the map; it is about 200 km south of the Maipo River.

Chromosomal data
Standard karyotypes were obtained from bone marrow, liver, spleen and testes for a subset of specimens collected in each locality following the colchicine-hypotonic pretreated air-drying treatment (Evans et al. 1964), and stained with Giemsa and classified in accordance to a chromosomal race.

Characters
We scored 40 meristic scale characters on each individual.Many typical scale counts do not follow a normal distribution or are nearly invariant (Sites 1982, Lamborot & Eaton 1992); we include here only those 28 characters which were variable and approximately normal.The characters used were the following: CORA or Supraorbital semicircle: number of circumorbital scales separating the enlarged supraocular from median head scales; CORB: number of circumorbital scales plus the scales between the superciliaries and supraorbital; SOR: all scales inside CORB, within the semicircle of circumorbital scales; SCI or superciliaries: row of enlarged scales lying in the border of the upper eyelid; TEM or temporal scales: number of scales between the posterior margin of the eye and the middle of the ear; SAP: number of scales bordering the parietal; POSPA: number of postparietals; FRON: frontal scale or number of scales in the area of the frontal scale; SAF: number of scales bordering the frontal(s); POSFR: number of postfrontal (s); LORA: scales in the loreal region between canthals, supralabials and subocular; LORB: LORA scales plus the supralabials; LASUP2A: row of scales over the first row of labials, from the rostral to the level of the posterior end of the enlarged subocular; LASUP2B: second row of scales from the rostral to the level of the labial commissure; LAINFA: number of enlarged infralabial scales behind the mental; AURS: number of enlarged scales protuding on the anterior edge of the right side ear opening; SARO: number of scales bordering the rostral; SANA: number of scales bordering the nasal; SBRF: number of scales in the middle way between the rostral and the frontal; GUS: row of scales across the gular region, between each anterior margin of the ear openings; DOS: number of scales in the vertebral line from the posterior margin of the head scales to the level of the genital ridge; VES: number of ventral scales from the posterior margin of the mental scale straight back to the base of the genital ridge; SAB: number of scales around the midbody; DORCA: number of mid-dorsal vertebral scales in a line equal to the length of the head; AVCO: number of scales around the 15th verticile of the tail counted from the level of the genital ridge; LAMA: number of subdigital lamellae on the fourth toe of the right forelimb; LAPA; number of subdigital lamellae on the fourth toe of the right hindlimb; APOR: number of preanal pores (only in males).In previous analyses, asymmetry was tested by measuring scale characters on both sides of the body in four populations.Since we found no appreciable evidence for asymmetry (Lamborot & Eaton 1992) we measured only the right side of the body for bilateral characters.
Relative variability for characters of males was determined as previously described (Eaton 1971, Lamborot & Eaton 1992).We also calculated the average of the coefficients of variation for purpose of comparison.

RESULTS
Although the means for most characters were not notably different among populations (Table 2), all 28 characters showed significant differences (analysis of variance, P < 0.05), which justifies their use in multivariate analysis.Previous morphological analyses demonstrated a certain degree of difference between characters means of males and females from the same population, however, the differences were small enough that the patterns revealed by factor analysis were not distorted by combining data (Lamborot & Eaton 1992, 1997).We therefore combined the data of the females and males for most analyses to increase sample sizes.The number of anal pores (APOR) is the character which showed the greatest differences among populations (Table 2), this trait can only be scored in males.In order to include this character in the analyses which combined males and females, we used the mean of APOR in males as the "population" sample mean.However, there were no males in the sample from Cabrería (sample 1), thus it was necessary to exclude this locality from the analyses which used APOR.The affinities of Cabrería were determined in an analysis using only females (see below).

RIVERINE BARRIER TO L. MONTICOLA CHROMOSOMAL RACES IN CHILE
t h e f i r s t t h r e e f a c t o r s g e n e r a l l y g r o u p e d populations according to chromosome race (Fig. 4), and separates the "Southern, 2n=34" and the "Northern, 2n=38-40" plus the" MF, 2n=42-44".One exception is sample Nº13 (Río Colorado Norte) which clusters with those of the Multiple Fission race, and two samples of the Northern race (Yeso Norte and San Gabriel) that appear the most different.While the separation of the races is evident, the results are not completely satisfying, as the algorithm used cannot show the position of intermediate populations.
Based upon the results of factor analysis, we combined data for populations of the same chromosomal race which were geographical neighbors and had similar values for the first two factors: the "Southern" race Cantillana from the c o a s t ; Y e s o S u r ( a z o n e o f s e c o n d a r y hybridization); San Gabriel plus Yeso Norte because they are different from the other "Northern" race samples; Cuesta Chacabuco from the Transverse Range; Colorado Norte and Colorado Sur from the Andes.We included APOR and used both males and female in order to have 28 characters and nine groups (Fig. 5).
Relative variability for all characters for each sample for males and females separately or for males plus females were somewhat contradictory, the greatest values were Cantillana ( 14) and Los Queñes (15), both belonging to the "Southern,

Origin of the chromosomal races
Since actually, and based on cytogenetic studies (Lamborot 1993(Lamborot , 2001)), we do not have evidence for chromosome introgression from one race into the next, (except in the narrow hybrid zones) and because the chromosomal evolution has probably occurred via successive chromosomal rearrangements, producing a linear series of chromosomal races, whose complexity increases south to north and based upon previous results (Lamborot & Eaton 1997, Lamborot 2001), we suggested that the "Northern, 2n=38-40" race originated from the "Southern, 2n=34" race in the Coastal Range, crossed to the Andes using the Transversal Range (sample 8 or C) as a bridge, and only later reached the Andes north of the Maipo River (Fig. 4).This would explain the greater morphological differences between the races in the Andes (D = San Gabriel and Yeso Norte) that are separated by the Maipo River in 200 m.South of the Maipo River and south of the Yeso River, all the L. monticola populations have 2n < 34 chromosomes, while north of the river, all populations have 2n > 38 chromosomes, which suggests that the "Northern, 2n=38-40" race was either produced in the founding population, or once produced, it replaced the other "Southern, 2n=34" race populations north of the Maipo River.
The Aconcagua River is also a biogeographical barrier that separates the "Northern, 2n=38-40" race from the "Multiple Fision" race.Later, the "Multiple Fision" race (G) originated from the "Northern" race.Some of the chromosomal changes may have occurred in the Andes, because samples from north and south of the Colorado River, one of the tributaries of the Aconcagua River, present intermediate morphological values between these races and appear to be intermediate in our preliminary chromosomal survey; they are polymorphic for a pericentric inversion and an enlarged chromosome, two of the five rearrangements which are present in the "Multiple Fision" race (Lamborot 1998, M. Lamborot unpublished data).Also the Colorado River appears to separate the samples from Colorado Norte (F) and Colorado Sur (E), the former being more closely related in morphology and geographic proximity to the "Multiple Fision" race.According to our hypothesis, the Colorado River samples should represent transition populations, rather than a zone of secondary contact.In fact our preliminary chromosome survey indicates that both population samples of the Colorado River correspond to a new chromosomal race with the same diploid number of those from the "Northern, 2n=38-40" race, but with two out of five new chromosomal mutations that are present in those of the "Multiple Fission, 2n=42-44" race.We plan to use more sensitive molecular genetic markers to provide further evidence for the proposed scheme.The greater degree of differences associated with the Maipo River and the sequence of chromosomal changes argues that this barrier was crossed first, in other words that this species was gradually expanding to the north.What is more remarkable is that in each case, the crossing of a river barrier has been accompanied by chromosomal changes in the founding population or in a new population which then replaced the older one.These results provide evidence that both rivers have been important in the chromosomal and morphological differentiation by interrupting gene flow, and by limiting the reexpansion of spatially disjunct differentiates.We hypothesize that the other new polymorphic fissions that characterize the "Multiple Fision" race (Lamborot 1998) could have arisen in the Coast Range in a similar way as the origin of the "Northern" race that escaped the glacial action.
The association between chromosomalmorphological races and river barriers may be explained in part by historical factors.Geological data for this region demonstrate that Pleistocene glaciation was extensive.In the next-to-last episode, the glacier of the Maipo Valley was particularly well developed (Brüggen 1950, Vuilleumier 1971).In the Aconcagua Valley, Caviedes (1972) considers that similar phenomena occurred, the three major Quaternary glaciations are represented in this river system and possibly a fourth glaciation which reached 1.100 m, in the deep valley of the Colorado River.The middle course of the Aconcagua River crosses intermontane basins and cuts though the ridges of the Coastal Range.The chronology accepted by Caviedes (1972), allows the presumption that the glaciations of the middle Chilean Andes occurred "in phase" with those of the Northern Hemisphere, but never reached a similar intensity.Morraines are found in the central valley and on the eastern slopes of the Coastal Range, but not on the western slopes of the latter (Formas 1979). However, Heusser (1966) indicated that the Coast Range escaped the action of glaciers, from which we conclude that glacial tongues may well have acted as greater barriers for L. monticola in the Andes than in the Coastal Range for both the Maipo and the Aconcagua rivers.
Summarizing, our results provide evidence that the Aconcagua River (32º 52' S) as well as the Maipo and Yeso rivers (33º 46' S) have been important in the differentiation of populations of L. monticola.This is a good example of parapatric differentiation (Pounds & Jackson 1981) produced by riverine barriers.Both rivers present similar Pleistocenic historical events (Brüggen 1950, Caviedes 1972).
These rivers have acted as barrier more strongly near the headwaters of the Aconcagua River than its mouth, the former represented by the Colorado River samples.We don't know if the other chromosome fission polymorphism mutations of the "Multiple Fission" race were originated from the "Northern" race at the Coastal Range and then hybridized (Fig. 4).These hypotheses should be further analyzed utilizing more sensitive (genetic) markers such as allozymes or mtDNA, in order to evaluate past patterns of gene flow.

Fig. 1 :
Fig. 1: Map of central Chile showing locations of 18 sampling sites of Liolaemus monticola (see Table1) in the Andes, Coastal and Transversal ranges.Numbers enclosed in triangles represent sampling localities of the "Multiple Fission, 2n=42-44" race; those enclosed in circles represent sampling localities of the "Northern, 2n=38-40" race; those enclosed in double circles represent sampling localities of the "Northern modified 1, 2n=38-40" race, north and south the Colorado River, an affluent of the Aconcagua River; those enclosed in squares are localities of the "Southern, 2n=34" race; the circle and square together represents the zone of secondary contact.The sampling locality of Los Queñes (15) is not shown on the map; it is about 200 km south of the Maipo River.

Fig. 4 :
Fig. 4: Sokal's distance phenogram resulting from UPGMA clustering of the first two principal components of 29 meristic characters measured in 18 samples of Liolaemus monticola from the middle Chile.Numbers at the ends of branches correspond to the sampling localities.The symbols correspond to the chromosomal races.

TABLE 1
Localities, chromosomal races and sample sizes of the Liolaemus monticola monticola populations used in this study.Sample numbers correspond to those of figuresLocalidades, razas cromosómicas y tamaño de las muestras de las poblaciones de Liolaemus monticola monticola usadas en este estudio.Los números de las muestras corresponden a la de las figuras

TABLE 2
Mean values for 28 meristic characters in samples of 19 populations of Liolaemus monticola monticola from central Chile.For character and locality explanations, see Material and MethodsValores promedios para 28 caracteres merísticos en muestras de 19 poblaciones de Liolaemus monticola monticola de Chile central.Ver explicación de los caracteres y de las