BiologicalJournal ofthe Limean Society (ZOOO), 69: 263-281. With 4 fiLgures
doi:10.1006/bij1.1999.0367, available online at http://www.idealibrary.com on
@
IDfbl
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Speciation and adaptive radiation of
subterranean mole rats, Spalax ehrenbergi
superspecies, in Jordan
EVIATAR NEVO'*, ELENA IVANITSKAYA', MARIA GRACIA
FILIPPUCC12AND AVIGDOR BEILES'
'Institute of Evolution, Universip of Ha@, Ha@ 31905, Israel, 2Dyjartimentodi Biologia,
I1 Universitu di Roma 'Zr Vqata', l4a 0. Raimondo 00173, Roma, Itah
Received 18 December 1998; acceptedfor publication 5 April 1999
The major initial mechanism of speciation in subterranean blind mole rats, Spalacidae, is
chromosomal, primarily through Robertsonian rearrangements. Here we highlight another
scenario of chromosomal rearrangement leading to ecological speciation and adaptive
radiation apparently initiated by pericentric inversions and genic divergence to different
ecologies in mole rats in Jordan. We analysed karyotype, allozyme, size and ecological
diversity across the range of mole rats in Jordan from mesic Irbid in the north to xeric Wadi
Musa (Petra region) in the south, a transect of 250km. We examined mole rats for
chromosome (N= 71), size (N= 76), and allozyme (N=67) diversities, encoded by 32 loci, in
12 populations of the Spalax ehrenbep'superspeciesinJordan. By a combination of chromosome
morphology, genetic distance, body size and ecogeography, we identified four new putative
biological species. All species (except two animals in Madaba) share 2n=60 but vary in
chromosome morphology, caused by pericentric inversions and/or centromeric shifts. The
'north Moav' species is karyotypically polymorphic for 2n (2n=60; including locally also two
animals with 2n = 62). The distribution of the four species is associated with ecogeographical
different domains and climatic diversity. Genetic diversity indices were low, but like chromosome arms (NFa) were positively correlated with aridity stress. Discriminant analysis
correctly classified 91% of the individuals into the four species utilizing combinatonally
chromosome, allozyme and size diversities. It is hypothesized that mole rat evolution
underground is intimately associated with climatic diversity stress above ground.
0 2000 The Linnean Society of London
ADDITIONAL KEY WORDS:-blind mole rats -Jordan - allozymes
pericentric inversions - speciation - adaptation - ecogeography.
~
karyotypes
-
CONTENTS
Introduction . . . . . . . . . . . . . . . . . . . . . . .
The ecological theatre . . . . . . . . . . . . . . . . . .
Material and methods . . . . . . . . . . . . . . . . . . .
Results and Discussion . . . . . . . . . . . . . . . . . . .
Cytotype diversity . . . . . . . . . . . . . . . . . . .
Ecological correlates of cytotypes of mole rats in Jordan . . . . . . .
264
265
266
266
266
272
* Corresponding author. E-mail: [email protected]
0024-4066/00/020263+19 $35.00/0
263
0 2000 The Linnean Society of London
264
E. NE\’O E T A L
Allozyrne diversity . . . . . . . . . . . . . . . .
Putative four species of Spalax ehrenbegi in Jordan . . . . . .
Discriminant analysis . . . . . . . . . . . . . . .
Dating the origin of the species in the Spalax ehrenbergi superspecies
. . . . . . . . . . . .
Environmental differentiation
Conclusions . . . . . . . . . . . . . . . . . . . . .
A new scenario of speciation in Jordanian Spalax . . . . . .
Acknowledgements
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References . . . . . . . . . . . . . . . . . . . . .
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1NTROI)UCTION
The Spalacidae are Southeast European and East Mediterranian blind rodents,
highly adapted for life underground (Nevo, 1991, 1995, 1999). Their taxonomy,
biogeography, palaeontology, evolutionary origins, chromosomal evolution, population biology and species concept were reviewed (Savit & Nevo, 1990). Their
taxonomy needs a modern revision including chromosomal and molecular-genetic
data as well as morphology, physiology, and behaviour. The Spalacidae originated
probably from a muroid-cricetoid stock in Asia Minor in Oligocene times and
adaptively radiated by chromosomal speciation. They budded species with higher
diploid chromosome number (2n) into three major directions: (i) Balkans, (ii) steppic
Ukraine and Russia, and (iii) Near East and North Africa, and a minor one (it,)
central Anatolia (Nevo et al., 1994b, 1995; Savit & Nevo, 1990).
The classical species Spalax ehrenbergi has been studied multidisciplinarily in Israel
during the last four decades as an evolutionary model of both speciation and
adaptation (Nevo, 1991, 1999). It is a chromosomal superspecies complex involving
four sibling species (2n=52, 54, 58 and 60), each adapted to a particular climatic
regime. Though not yet formally named, their status as a distinct biological
species is justified due to the following criteria. (i) The four species are distributed
parapatrically, separated by increasingly narrowing hybrid zones northward
(2.80-0.32 km), but without chromosomal introgression. (ii) Multiple adaptive
systems (genetical, ecological, biochemical, morphological, physiological and behavioural) characterize each species, adapting it to its unique ecogeographical
climatic regime (Nevo, 1991). Postmating chromosomal incompatibilities with partial
infertility, and premating ethological mechanisms (olfaction, vocalization, seismic,
aggression, mate-choice, bacular structure and function, and possibly tactile and
gustatory mechanisms) reproductively isolate the species (Nevo, 1990), complementing the chromosomal incompatibilities which initiated speciation. Genetically,
the most remarkable spalacid phenomenon in Israel is the positive correlation of
diploid numbers, 2n, and heterozygosity, H, with increasing aridity and climatic
unpredictability. 2n increased by Robertsonian rearrangements towards the southern
(2n=52+58-+60) and eastern (2n=52+54) Negev and Syrian deserts, respectively.
We tested the evolutionary mode of positive association of 2 n and H with aridity
stress in mole rats on a 30-time larger scale in Asia Minor (Nevo et al., 1994b, 1995).
We found extensive Robertsonian chromosomal speciation in S. leucodon (2n = 38,
40, 50, 54, 60 and 62) and in 5’. ehrenbergi (2n = 52, 56 and 58) and 2n = 54 (Yuksel
8: GulkaC, 1992), presumably representing 14 to >20 additional biological species.
Both 2n and H were positively correlated with aridity stress, increasing centripetally
from the periphery toward geologically young, arid, and climatically unpredictable
SPECIATION OF MOLE RATS IN JORDAN
265
central Anatolia. Chromosomal, allozymic and ecological diversities supported the
hypothesized biological species status of most tested populations, extrapolated on
the basis of the Israeli model. The Israeli and Turkish models involve ecological
speciation through Robertsonian chromosomal rearrangements. Both in Israel and
Turkey pencentric inversions in the Spalacidae were documented and considered
by us hitherto only as local adaptation devices.
We have suggested that Robertsonian fissions are the main mechanism of speciation
in Spalax (Wahrman et al., 1969a,b, 1985; Nevo, 1979, 1991, 1995; SaviC & Nevo,
1990). Such mechanism provides both postmating reproductive isolation, as well as
adaptive higher levels of recombination with increased 2n (Nevo, 1991; Nevo et al.,
1994b). More than 30 cytotypes (2n = 3%-62, NFa = 72-1 20), earlier represented by
eight classical species, occurring primarily dopatrically or parapatrically were known
in 1990 (Savib & Nevo, 1990). We also described another new species from Africa
displaying 2n= 60 (Lay & Nadler, 1972),based on chromosome morphology, ecology
and behaviour (Nevo et al., 1991). Recently, three new cytotypes of Spalax leucodon
superspecies were determined from Turkey, having 2n=52, 56 and 5%(Sozen &
Kivanq, 1998a,b). In all, more than 50 cytotypes have been described in the
Spalacidae, most of which are good biological species displaying explosive ecological
adaptive radiation into steppic environments.
For many years we wondered, but could not explore, what was the mole rat
pattern of chromosomal evolution in the highlands, east of the Jordan-Arava rift
valley, in the Kingdom ofJordan. Peace with Jordan opened for us the possibility
to examine the evolution of mole rats of the Spalax ehrenbergi superspecies in Jordan,
thus studying a 'missing link' in the stepwise colonization of S. ehrenbw' from southern
Turkey through the Near East to North Africa. Here we describe four new putative
species of the S. ehrenbmgi superspecies in Jordan, characterized by chromosome
morphology, size, genetic distance, and ecogeography. This substantiated our hypothesis that the evolutionary process in Spalax is driven by natural selection in
accordance with aridity stress. Furthermore, we showed that the mechanism of
speciation in Jordanian mole rats is novel for Spalacidae. It was initiated not as
usual in the Spalacidae primarily by Robertsonian chromosome fission (Nevo, 1991;
Wahrman et al., 1969a,b, 1985;Nevo et al., 1994b, 1995)but by pericentric inversions,
or centromeric shifts (i.e. changes in chromosome morphology) and/or genic
divergence without change in diploid chromosome number (2n).
The ecological theatre
Jordan consists of 90 000 km2divided into three longitudinal zones: (i) the rift
valley extending 370km from the Lake of Tiberias in the North to the Gulf of
Aqaba in the South, ranging in width 9-25 km. (ii) The Eastern Mountain ranges,
extending 350 km in length and covering over 2 1 000 km2from the north down to
the south in Ras En-Naqb, comprising the Mountains of Gilead, Ammon, Moav
and Edom, ranging in width 30-50 km,and cut by major east-west rivers, Yarmuk,
Zarqa (= Yabok), Mujib ( =Arnon) and Hasa ( =Zered), and (iii) the Eastern Desert,
extending into the Syrian-Arabian Deserts.
The Spalax ehrenbeg$ superspecies extends in Jordan primarily in the mountain
ranges from Gilead to the southern Edom mountains, the latter rising up to 1700 m
266
E. NEL’O E T A L
(Fig. 1). Annual rainfall varies across Jordan from 600mm at Ishtafina in Ajloun
area south of Irbid, to 50 mm in the Arava Valley (Long, 1957; Atlas ofIsrael, 1970;
Al-Eisawi, 1985). Mean annual minimum temperature ranges between 5-20°C, and
mean annual maximum between 20-30°C. Summer temperatures reach 40°C in
the Arava rift valley (Long, 1957; Al-Eisawi, 1985). Generally, rainfall decreases
and temperature increases southwards and eastwards, with much spatiotemporal
variation, from 600 mm in Gilead and Ajloun to 300 mm in Moav to 100-300 mm
in Edom, and 50 mm in the Southern Arava Valley near Aqaba. Biogeographically,
Jordan consists of eight or nine bioclimatic regions (Long, 1957; Al-Eisawi, 1985),
and three major phytogeographical regions (Atlas of Israel, 1970; Zohary, 1973;
Al-Eisawi, 1985). These involve the Mediterranean (subhumid), Irano-Turanian
(semiarid) and Saharo-Arabian (arid) regions.
MATERIAL AND METHODS
Subterranean blind mole rats of the Spalax ehrenbergi superspecies occur in Jordan
primarily in the subhumid, semiarid and arid regions of the eastern Mountains,
plateaus and their margins, ranging from Irbid in the north to Ras El Naqeb in the
south (Fig. 1). As in Israel, Spalax does not penetrate into regions with less than
100 mm annual rainfall, the eastern Jordan deserts, or the Arava valley. We analysed
weight (N= 76), karyotype (N= 7 l), and allozymic (N= 67) diversities (at 32 gene
loci), in subterranean mole rats of the Spalax ehrenbelgi superspecies from 12 localities
across the Kingdom of Jordan. Collections were conducted in two field excursions
during January and March 1996. Their ecogeographical background and results on
weight, karyotypes, and allozymes are given in Table 1. The collecting sites,
superimposed on the rainfall isohyetes, and the major mountain ranges with the
east-west rivers cutting the ranges, appear in Figure 1.
Climatic and geological data were taken from Al-Eisawi (1985) and the Atlas of
h a e l (1970). The ecogeographical data (climate, soil, etc.) appear in Table I .
Chromosomal techniques (G, C banding and NOR) appear in Wahrman et al.
(1 985) and Ivanitskaya & Nevo (1 998), and those of allozymes in Nevo et al. (19944.
The high number of loci analysed ensured good estimates of genetic distances among
populations in spite of the relatively small sample size of some populations. Values
of observed heterozygosity per individual, H, genic diversity, He, and genetic
distance, D,are therefore reliable with a reasonable margin of precision (Nei, 1978).
Furthermore, because we earlier analysed chromosome and allozyme evolution in
Spalax (Nevo et al., 199413, 1995), we preferred sampling more localities across Jordan
than larger numbers of animals in each locality. Specimens are deposited at the
Institute of Evolution, University of Haifa.
RESULTS AND DISCUSSION
Cytoppe diversip
We have found six different karyotypes, five with 2n = 60 and NFa = 68, 70, 7 2 ,
74 and one with 2n=62 (only two animals) and NFa=70 in the Spalax efzrenbergi
SPECIATION OF MOLE RATS IN JORDAN
261
Figure 1. Sampling localities of the Spalax ehrenbq’ superspecies in Jordan, superimposed on an annual
rainfall isohyete map. Note that rainfall decreases both southward and eastward. Localities: 1 - Irbid,
6 km S, Gilead Mountains; 2 - Zarqa, 10 km northwest Ammon Mountains; 3 Naur, 25 km North
of Madaba, Ammon Mountains; 4 Mount Nebo, 5 km northwest Madaba, Northern Moav Mountains;
5 Madaba S, 6 km South, Northern Moav Mountains; 6 - Madaba E, Jizah, 10 km East of Madaba,
North Moav Mountains; 7 - Dhiban, 5 km North of Wadi Mujib (Nahal Arnon), Northern Moav
Mountains; 8 - Ariha, 5km South of Wadi Mujib, Southern Moav Mountains; 9 - Karak, 3 k m
Northeast, Southern Moav Mountains; 10 - Mazar, lOkm North of Wadi Hasa, Southern Moav
Mountains; 11 - Tafila, 5 km North, Edom Mountains; 12 - Wadi Musa, 7 km Northeast, Edom
Mountains. Mountains from North to South: Gilead, Ammon, Moav and Edom.
~
~
~
268
ns: M=hfale; F or Fern= Frmale; Tot =Total.
al variables:
?n=diploid number;
NFa=Autosomal number of chromosome arms;
Acr = Number of acrocentric chromosomes;
Biar=Number of biarmed chromosomes. In parenthesis thr number of small biarmed chromosomes;
NOR=Numhers o i N O R bearing chromosomes;
Pair# morph. =Morphology of chromosomes number 1, 7, 26, 29:
A =Acrocmtric;
M =hlrtacentric;
S =Subreloc~ntric;
S-l =Short arm usually larger than the short arm of thr x-chromosome and c-negatiLje;
S-2 =Short arm with the length like the short arm of thr x-chromosome and r-positive;
S-3=Shon arm shorter than the shan arm of the x-chromosome and c-positive;
S-4=Short a m very short, almost invisible.
A = Mean number of alleles per locus;
ices:
H=Mean heterazygosity per individual;
He =Genic diversity;
P-5% = Proportion of polymorphic loci, 5% criterion;
erorlimatir variahlrs:
raphical
Ln =longitude, in decimals
Lat =latitude, in derimals
Alt=altiNde, in meters
xrarurc:
T a =mean hottest month temprraturc (“C)
Tj = mean coldest month temperature (“C)
Tav = mean T a and Tj (“C)
Td=seasonal temperature diiferencc (“C)
r availability:
Rn = mean annual rainfall, in mm
Rrl =mean number of rainy days
Q= 1000 x Rn/(Tav x Td), Tav in Kelvin
Sod = soil type: TR = terra rossa
hir :
Rm=rendzina
R-B = Brown rendzina
limatic region according to Long (1957): 1 =Mediterranean, sub humid
2 =Mediterranean, semi arid
3 = IranwTuranean, arid cold
4= Iran-Turanean, arid warm
enzymes used in electrophoresis:
-glyc~rophosphate dehydragenaw (E.C. 1.I.1.8);
enosine deaminase (E.C. 3.5.4.4);
rohol dfhydrogenases (E.C. 1.1.1.1), not used in analysis, hrcause of missing data. Was monomorphlc;
mylate kinase (E.C. 2.7.4.3);
olaae (E,.C. 4.1.2.13);
e a t i n ekinas? (E.C. 2.7.3.2);
sterase (E.C. 3.1. I.I);
umaras~(E.S. 4.2.1.2);
artate transaminase (E.C. 2.6.1.1), two IWI;
lyceraldehyde-3-phosphate dehydrogenasr (E.C. 1.2.1.l2);
lucose-6-phosphatc dehydrogenase (E.C. 1.1.1.49);
exokinases (E.C. 2.7.1.1);
ophcnnl oxidase (E.C. I. 15.1. I),
iuate dehydrogenase (E.C. 1.1.1.42), two loci;
cyl aminopeptidase (E.C. 3.4. I 12);
tate dehydrogenase (E.C. 1.1.1.27), two loci;
alaie dehydrogenase (E.C. 1.1.1.37), two lor<
lic rnzvme (E.C. 1.1.1.40), two loci;
annosrphasphate isomerase (E.C. 5.3.1.8);
leosidc phospharilaw (E.C. 2.4.2.1);
cose-fi-phosphateisomerases (E.C. 5.3.1.9);
ospho~l:luromutasp(E.C. 2.7.5.1 now 5.4.2.2), two loci;
eral protein,, three loci;
-Phorpho~Iuranate dehydrogrnasrs (E.C. 1. I . 1.44);
bitol drhydrogenasr (E.C. 1 , l . I . 14)
Pm
E. NEVO ETilL.
270
C
f Pt-t
1
-r;-*;
26
29
30
Figure 2. Variable chromosomes from haploid sets ofJordanian Spalax from Irbid (NFa = 74) Madaha
N , Naur (NFa=72), Madaba S (NFa=68) and Tafila (NFa=70). (A) Conventional staining, (B) Cbanding, (C) G-banding patterns. The chromosome numbering corresponds to fig. 4 in Ivanitskaya &
Nevo (1998). Chromosome no. 30 (the last in the right hand column) is an 'additional' acrocentric
chromosome that appeared only in 2 animals from the third cytotype, type B. The figure is from a
male karyotype from Madaba S (2n=62, NFa=70). Dashes indicate the centromere positions in Gbanded chromosomes.
superspecies in Jordan. Detailed description of chromosomal morphology, G- and
C-banding patterns of all karyotypes appears in Ivanitskaya & Nevo (1 998). Here
we only describe and illustrate the basic karyotypic differences between differing
cytotypes (Table 1, Fig. 2), coupled (see later) with allozyme diversity, size, and
ecogeography as the combinatorial basis of speciation of mole rats in Jordan.
The first cytotype with the highest number of autosomal arms in Jordanian Sflalux
(NFa = 74) was found in Irbid and Azzarqa (= Zarqa) (populations nos. 1 and 2). It
differed from other cytotypes by the biarmed form of the 7th and 29th autosomal
pairs. The first pair is subtelocentric with length variable, C-negative short arms,
and heterochromatic pericentromeric block in the long arms (Table 1, Fig. 2). The
second cytotype with NFa= 72 was found in Naur, North of Madaba (population
no. 3) and in Mt. Neb0 (population no. 4). The main characteristic of this cytotype
was the biarmed condition of the pair no. 26. The first pair is subtelocentric with
length variable, C-positive short arms (Table 1, Fig. 2). The third cytotype was
found in Madaba S, Jizah (Madaba E) and Dhiban (populations nos. 5, 6 and 7)
and includes two types. Type A had the lowest number of autosomal arms (NFa=
68) in Jordanian mole rats. Type B was found in two animals in the Madaba S
population, had 2n=62 (NFa= 70), the highest 2n of mole rats so far found in the
entire Near East. The increasing 2n in this karyotype is caused by an additional
pair (no. 30) of small acrocentric chromosomes (Fig. 2 and Table 1). The main
SPECIATION OF MOLE RATS IN JORDAN
27 1
distinguishing feature of this cytotype is the acrocentric morphology of the first pair
in both types A and B (Fig. 2 and Table 1). Partial deletion of the short arms
followed by pericentric inversion is responsible for the acrocentric morphology of
this pair. These types of rearrangements were supported by G-banding methods
(Fig. 2, and Ivanitskaya & Nevo, 1998).
It is difficult to explain the origin of the smallest ‘additional’ acrocentric chromosomes in the S. ehrenbqi karyotype. Possible candidates for their ancestries are
the short arms of the first variant pair (fission rearrangement) and a small pair of
acrocentric autosomes (duplication). We can exclude recent duplication because of
the size and G-banding pattern of the additional pair. The recent fission of the first
pair is unlikely also due to the fact that the morphology of the first pair in 17
individuals of this cytotype is acrocentric and originated by inversion. Furthermore,
the ‘additional’ pair has G-positive centromere region. By contrast, the centromere
region is G-negative in all subtelocentric first autosomes.
The fourth cytotype with 2n = 60 and NFa = 70 was found in Mt. Nebo, Ariha,
Karak, Mazar, Tafila and Wadi Musa. This cytotype is largely geographically
separated from the third cytotype by the huge canyon of Wadi Mujib (Nahal Arnon),
although it does appear in two animals north of the canyon in the Mt. Neb0
karyotypically polymorphic population. It differed from the third cytotype by the
subtelocentric morphology of the first pair. Geographical variability of the short
arm length was revealed in this cytotype (Table 1). The gigantic canyon of Wadi
Hasa (Nahd Zered) separated the fourth cytotype with NFa = 70 into two groups:
(i) Ariha, Karak and Mazar, populations nos. 8-10, ranging from the canyon of
Wadi Hasa northwards to the canyon of Wadi Mujib, characterized by almost
invisible short arms of the first pair, and (ii) Tafila and Wadi Musa, nos. 11 and 12,
ranging from Wadi Hasa southwards, characterized by visible short arms of the first
pair (Fig. 2). Among the 12 investigated populations only two (Madaba S and Mt.
Nebo) were karyotypically polymorphic (Table 1).
AU examined cytotypes had a similar distribution of heterochromatin material,
except the chromosomes shown in Figure 2. Acrocentric chromosomes had more
or less large blocks of pericentromeric heterochromatin. Biarmed autosomes, excluding the h s t pair, were C-negative. Variable autosomes (pairs 7, 26 and 29) were
C-negative at biarmed forms, and had centromeric C-blocks at acrocentric forms
without observed intrapopulation variability. The greatest intopopulation differentiation in C-banding patterns were revealed in the first pair (Fig. 2, and Jvanitskaya
& Nevo, 1998)We have discussed the variable short arms of this pair in cytotypes with NFa=
74, 72 and 70, which had different C-banding patterns ranging from C-negative to
C-positive of the whole or part of the arm. G-staining revealed in the short arm of
the first chromosome euchromatic variation in addition to the heterochromatin
variation described earlier (Fig. 2). Thus, the intercytotype variation is expressed in
both types of chromatin. The intrucytotype variation is much smaller than the
intercytotype variation. The acrocentric first chromosome of FNa =68 showed no
morphological variation in all examined animals. We have shown that the acrocentric
first chromosome of NFa = 68 evolved by a deletion of part of the short arm followed
by a pericentric inversion (Jvanitskaya & Nevo, 1998).
The number of nucleolar organizing regions in Jordanian Spalax is associated with
the morphology of the first chromosome pair (Ivanitskaya & Nevo, 1998). Cytotypes
with subtelocentric chromosomes of this pair (2n=60 and NFa=70, 72, 74) had
272
E. NEVO B 7 4 L .
two NOR bearing pairs (telomeric regions of the first and fifth chromosomes); and
cytotype with NFa = 68 (including the karyotype with additional two chromosomes)
with the acrocentric first pair had only one (5th) NOR bearing pair. It is worth
noting that the variability of the short arm length in the first chromosome pair
(especially in the two southern groups) does not affect the presence of NORs. The
change of the morphology of chromosome no. 1 by pericentric inversion (likely
followed by deletion) has led to deletion of nucleolar sites of the cytotype 2n=62.
The main difference between the karyotypes in Jordan was caused by pericentric
inversion or centromeric shifts in four chromosomal pairs (Fig. 2). Two of these
pairs (26 and 29) belong to Group C, the group of inversion chromosomes, which
was not considered responsible for speciation in the S. ehrenbqi superspecies in Israel
(see Wahrman et al., 1969a,b, 1985). Pericentric inversions of Group C do not
increase diploid chromosome number but contribute to variation in NFa (72-80) in
Israel, both within and between the four chromosomal species of mole rats (2n=
52, 54, 58 and 60). They have been considered adaptive chromosomal rearrangements, but have not hitherto been considered as initiators of speciation in
Spalax (Wahrman et al., 1969a,b, 1985). Changes of centromere position in the first
and seventh pairs of chromosomes from Group A (“Unchanged chromosomes”,
Wahrman et al., 1985) are the principal rearrangements that separate Israeli 2 n =
60 and Jordanian 2n = 60, which are also separated allozymically (see below).
Conventionallystained karyotypes of Israeli 2n = 60 with NFa = 72 and theJordanian
cytotype with NFa = 72 from population no. 3 (Madaba =Naur) seem identical, but
G-banding showed different position of centromeres in one chromosome pair, no.
7. Clearly, Israeli chromosomal species with 2 n = 52, 54 and 58 differ fromJordanian
karyotypes, as well as from Israeli 2 n = 60 by a series of Robertsonian rearrangements.
The first cytotype from Irbid (2n=60, NFa=74) is geographically close to the
Afiq population in the southern Golan Heights with a karyotype of 2n = 58, NFa =
72, differing by one Robertsonian rearrangement and one pericentric inversion (the
present study and Wahrman et al., 1985). Both cytotypes are separated by the
Yarmuk river, and the M q karyotype diverged from the main range of 2n=58 in
Israel and is apparently a Holocene colonizer within the arid southern Golan
Heights, isolated from the main range and contained in a triangle bounded by the
Yarmuk and Jordan rivers. Molecular genetic analyses suggest that it may be a new
speciation cradle (Nevo, 1991).
Ecological correlates of CytoQpes of mole rats in Jordan
The cytotypes of Jordanian mole rats are associated with distinct geographic and
climatic regions. The big east-west rivers or canyons in Jordan dividing the eastern
mountain range into Gilead, Ammon, Moav and Edom Mountains also largely
separate between the different main cytotypes (Fig. 1). The first cytotype is bounded
in the south by Nahr az Zarqa. The second cytotype is bounded in the south by
Wadi a1 Kafrayn. The third cytotypes are largely bounded in the south by the huge
canyon of Wadi a1 Mujib (=Arnon river). The two groups of the fourth cytotype
are separated by the huge Canyon of Wadi al Hasa (= Nahal Zered). Mole rats
with these karyotypes also inhabit different climatic regimes as follows: The first
cytotype (2n =60, NFa = 74) ranges primarily in a subhumid to semiarid climatic
SPECIATION OF MOLE RATS IN JORDAN
273
TASLE
2. Coefficients of genetic distance (0)
between the four species of Spalax ehmbqi in Jordan
No. of
populations
1
1. ‘Gilead’
1
-
2. ‘South Moav’
3
0.050
(0.0484.052)
0.001
(O.OOW.001)
3. ‘North Moav’
6
0.093
(0.084-0.107)
0.040
(0.0324.054)
0.009
(O.OOW.021)
4. ‘Edom’
2
0.102
(0.1014.103)
0.059
(0.054-0.064)
0.026
(0.014-0.047)
Species
2
3
4
0.006
(O.Oas-O.006)
regime, in the Gilead Mountains, extending into Ammon (the Zarqa population,
No. 2). The second cytotype (2n=60, NFa=70, 72) extends in a subhumid-cool
climate. The third cytotypes (primarily 2n=60, NFa=68-72) range in semiarid
warm climatic regime. The first group of the fourth cytotype ranges in an arid-warm
climatic regime. Finally, the second group of the fourth cytotype ranges in a
semiarid-arid cold climatic regime.
Are these cytotypes, underlying morphologically indistinguishablemole rats, good
biological sibling species? To answer this question we analysed the genetic distances
(D),
based on allozyme diversity, and compared them with Ds throughout the range
of the S. ehrenbqi superspecies in the Near East from South Turkey to Israel and
Egypt (see SaviC & Nevo, 1990, and Nevo, 1991 for the importance of allozyme
divergence in spalacid speciation).
Allogme diversip
We analysed 67 mole rats, collected in Jordan for allozyme diversity at 32 putative
gene loci (see enzymes at bottom of Table l), to assess the genetic differences
between populations. Average genetic diversity indices were low, as is true for
fossorial and subterranean mammals in general (Nevo et al., 1990; Nevo, 1999):
allele diversity A = 1.13, range 1.O-1.3, P= 0.099, range 0.OO-0.188; H = 0.030,
range 0.00-0.083; H,=0.031, range 0.00-0.067. The highest estimates of H and H,
were in Tafila and Dhiban in Edom and Moav Mountains, respectively. However,
the highest level of polymorphism was in Zarqa (Table 1). The average Nei genetic
distance (Nei, 1978) between the 12 populations of S. ehrenberg. in Jordan was D=
0.06, range 0.0W. 107. These estimates of genetic distances are critical for species
delimitation in the Spalacidae (SaviC & Nevo, 1990).
Futatioe four specks
of Spalax ehrenbergi in Jordan
Combining the foregoing description of the cytotypes (Table 1) with the genetic
distances of the 12 populations (Table 2 and Fig. 3), their body size and allopatric
ecogeographic regions, we obtained four putative biological species (Table 3 and
Fig. 4):‘Gilead’ (Irbid; 1); ‘North Moav’ [Naur =(Madaba N), Mt. Nebo, Madaba
E. NEVO ETAL.
274
Species
Populations
‘Gilead’[
Moav’
Irbid
Zarqa
Karak
Mazar
Mt. Neb0
‘North
Moav’ Madaba, S
Madaba, E
ArihaA
Tafila
‘Edom’
Wadi Musa
L
I
0.00
I
I
0.01 0.02
I
0.03
I
0.04
I
I
0.05 0.06
Genetic distance, D
I
I
I
I
0.07
0.08
0.09
0.10
Figure 3 . UPGMA dendrogram of 12 populations of the Spalwf ehrenbqi superspecies in Jordan based
on diversity and divergence of 32 allozyme gene loci. Note the division into four putative biological
sprcies ‘Gilead’, ‘South Moav’, ‘North Moav’ and ‘Edom’. Note that Madaba N is Naur and Madaba
E is Jizha.
S, Jizah (=Madaba E), Dihban and Ariha; 3-8)]; ‘South Moav’ (Zarqa, Karak and
Mazar; 2, 9, 10) and ‘Edom’ (Tafila and Wadi Musa; 11, 12). As seen in Figure 3,
the ‘Gilead’species is represented by the Irbid population only. The Zarqa population
displays the first cytotype (2n =60, FNa = 74) but is genetically distant from Irbid
(D=0.048) and is almost identical with Karak and Mazar populations (D=0.001),
which displays the first group of the fourth cytotype. Zarqa is located in a semiarid
eastern Irano-Turanian region similar ecologically (and genetically) to the ‘South
Moav’ Irano-Turanian populations of Karak and Mazar. Therefore, Zarqa appears
to belong to the ‘South Moav’ species, although it is distant geographically, but
similar ecologically, thereby forming a xeric geographic arc from Ammon to southern
Moav (Fig. 1).
The Ariha population, (no. 8) situated in South Moav, is closer to the five
populations of North Moav (D=O.001-0.021), north of Wadi Mujib, than to the
other south Moav populations. Its genetic distance from the other karyotypically
similar populations of South Moav (Karak and Mazar; 9, 10) is 0=0.033. Thus, it
was included with the karyotypically heterogeneous species of North Moav. The six
populations (3-8) of the ‘North Moav’ species are genetically similar (D=0.00-0.02 1).
However, they were karyotypically very heterogeneous, including two karyotypically
polymorphic populations (Mount Neb0 and Madaba S, the latter includes 2n = 60
and two animals with 2n = 62), the second cytotype in Naur (Madaba N), the typical
third cytotype in Dhiban, Madaba S and Jizah (Madaba E) and the fourth cytotype
in Ariha. This suggests that in Jordan CJytoppesalone are insuficient f o r species ident$cation.
Only combined with allozyme genetic distance, morphological (body size) and
ccological divergence they can contribute to delimitation of the four putative species
as is clearly revealed by discriminant analysis (Table 3; Fig. 4).
SPECIATION OF MOLE RATS IN JORDAN
275
TABLE
3. Discriminant analysis of 67 mole rats (Spalax ehrenberg’ superspecies) among four suggested
sibling species in Jordan, based on isozymes, karyotypic variables and body weight
(A) Chosen variables
Variables
Step
entered
in
Sdh
Mdh-1
Chr-29
fit-3
NOR
Chr-26
Got-1
Body weight
Wilks’
lambda
P
0.10175
0.01358
0.00525
0.00324
0.00254
0.00203
0.00165
0.00133
<0.00005
<0.00005
<0.00005
<0.00005
<0.00005
<0.00005
<0.00005
<0.00005
Label
Sorbitol dehydrogenase
Malate dehydrogenase- 1
Form of chromosome no. 29
Esterase-3
No. of NORs
Form of chromosome no. 26
Aspartate transaminase- 1
Standardized body weight
~
(B) Canonical discriminant function
Percent of
Function
Eigenvalue
variance
Canonical
correlation
After
function
Wilks’
lambda
Chisquare
~
P
df
~~
1
2
20.264
7.973
2.929
3
65.02
25.58
9.40
0.9762
0.9426
0.8634
0
1
2
0.0013
0.0284
0.2545
145.63
78.38
30.10
24
14
6
<0.00005
<0.00005
<0.00005
(C) Classification
Species
Actual
No. of
mole rats
‘Gilead’
1
7
‘North Moav’
2
35
‘South Moav’
3
17
1
8
5.9%
0
0.0%
‘Edam’
4
1
7
100.0%
0
0.0%
Predicted species location
2
3
0
0.0%
30
85.7%
0
0.O”h
0
0.0%
0
0.0%
5
14.3%
16
94.1Yo
0
0.0%
4
0
0.0%
0
0.0%
0
0.0%
8
100.0%
9 1.04% out of 67 mole rats correctly classified.
Dzscriminant anabsis
We ran a stepwise discriminant analysis (SPSS, 1990) on 67 mole rats that were
analysed allozymically. The program chose eight differentiating variables in the
following order: The isozyme loci Sdh, Mdh, morphology of chromosome 29, locus
Est-3, number of NORs, morphology of chromosome 26, the locus Got-1, and
standardized body weight (female weight x 1.312, the overall ratio of male/female
weight (Table 3 and Fig. 4). Two of the three significant functions (Table 3),
generated the pattern of the four putative biological species of mole rats of S.
ehrmbqi presented in Figure 4. The discriminant analysis succeeded to correctly
classify 91% of individuals into the four suggested species. Clearly, the species of
‘Gilead‘ and ‘Edom’ are widely separated. The separation of the ‘South Moav’ and
‘North Moav’ is less distinct and the third function (unseen in the figure) improves
the separation.
In summary, the four putative species of the S. ehrenbergi superspecies in Jordan
are based on a combination of different cytotypes, relatively large genetic distance
(D), body size diversity and ecogeographic distinct regions. The same combination
proved the best species discriminant in Israel (Nevo, 1991) and Turkey (Nevo et aL,
__
E. NEVO ETAL.
276
12.0
*
444
4.0 -
-4.0
4
4
I-
-8.01
-12.0
I
I
I
I
I
I
I
-12.0
-8.0
-4.0
0.0
CDFl
4.0
8.0
12.0
Figure 4. Stepwise discriminant analysis of mole rats in Jordan. Note the separation into four species:
1 - ‘Gilead’; 2 ‘North Moav’; 3 - ‘South Moav’; 4 - ‘Edom’. *Group centroid. The discrimination
is based on a combination of allozymes, chromosome morphology, and body size variables.
~
1994b, 1995).The average genetic distance between the four major biological species
in Jordan was D=0.062, range 0.026-0.100 (Table 2). These estimates are similar
to the genetic distances between the two northern (2n=52 and 54) and the two
southern (2n=58 and 60) chromosomal species (D=0.06) in Israel (Nevo, 1991).
Likewise, genetic distances in Jordan are similar to those in Israel (mean D = 0.0445;
range 0.003-0.093; Nevo et al., 1994a,b, Turkey (mean D = 0.135; range 0.052-0.263;
Nevo et al., 199413, 1995), between Israel and Egypt (mean D=0.044; range
0.001-0.184; Nevo et al., 1991).
All the four Israeli sibling species are considered on multidisciplinary grounds,
including pre- and postmating reproductive isolation mechanism, as good biological
species (Nevo, 1991). Likewise, genetic distance between other species of the S.
ehrenberg’ superspecies in the Near East (including Turkey, Israel and Egypt) ranges
between 0.003-0.269 (Nevo et al., 1994a).Consequently,we concluded that estimates
of genetic distances between the four species in Jordan justify us considering them
as good biological species. Further investigations are required to substantiate this
postulated speciation hypothesis, i.e. complete geographic mapping, unravelling
potential hybrid zones, and assessing pre- and postmating reproductive isolation, by
similar studies to those conducted in Israel (Nevo, 1991).
Dating the olligzn ofthe species in the Spalax ehrenbergi superspecies
The origin of the Israeli 2n = 60, the recent derivative of speciation in the Israeli
complex of four species (2n=52, 54, 58 and 60), was dated to 0.07-0.13 Mya
SPECIATION OF MOLE RATS IN JORDAN
277
(million years ago) both on allozyme and mtDNA grounds (Nevo et al., 1993) and
to 0.18-0.75 Mya by DNA-DNA hybridization (Catzeflis et al., 1989). Based on the
allozyme-derived genetic distances described in Jordan, the Jordanian 2n = 60
preceded the Israeli 2n=60. Moreover, chromosome pair No. 7 (Fig. 2) which is
invariably submetacentric in all Israeli species (including 2n= 60) is acmcentric in most
Jordanian karyotypes as is the case in Turkish S. ehrenberg’. The Israeli species have
17 autosomal pairs of group A, the ‘unchanged chromosomes’ (Wahrman et al.,
1985),whereas 10 pairs occur in group A in theJordanian karyotypes. TheJordanian
Spalax still retains its Turkish origins in the seventh chromosome pair. Nevertheless,
it is overall closer to the Israeli 2n=60 (from which it is still different) on both
allozymic and chromosomal grounds, described earlier. The complete elucidation
of karyotypic evolution in the S. ehrenberg. superspecies awaits the future analysis of
Syrian and Lebanese members of the S. ehrenbqi superspecies.
Enuironmenta 1 dgerentiation
The ecological basis of speciation in Jordan
Multiple regession. Is there any ecological basis to these four putative species?
T o answer this question we ran a multiple regression analysis of karyotypes on
ecogeography. We utilized in a multiple regression analysis the major differentiation
between cytotypes, i.e. the NFa (range 68-74, Table 1) as dependent variable and
used ecogeographical parameters as independent variables. The r2 =0.6 1, PC0.05
included three explanatory variables: longitude, seasonal temperature differences
and rain, i.e. predominantly climatic factors. This suggests that climatic variation and
stress may be linked to the differentiation of mole rat species in Jordan, as is the
case in Israel (Nevo, 1991) and Turkey (Nevo et al., 1994a,b, 1995).
Morphological d@irentiation
The mean weight of males and females in all 12 populations of S. ehrenbelgi
superspecies in Jordan was 115.6 g and 88.7 g respectively, significantly smaller than
the Israeli 2n=60 (males 129.5 g and females 106.8 9). Most .males in Jordanian
populations are smaller than the Israeli males in populations of 2n=60, excepting
Zarqa, Naur and Mount Neb0 populations. The average weight of females in all
12 Jordanian populations is lighter than Israeli females in 2n=60. This lighter
weight of Spalax in Jordan than in Israel follows Bergman’s Rule, i.e. decreasing
body size in inceasingly warmer environments (Nevo et al., 198613). Likewise, size
varies significantly (P<0.05) among the 12 populations.
The four putative mole rat sibling species inJordan are externally indistinguishable,
as is true for the Israeli four species. They can be identified at this stage only on
the combination of chromosome morphology, allozymes, and size diversities in four
ecogeographical domains, as described earlier. Notably, this was also true for the
four chromosomal species in Israel for which additional morphological speciesspecific characteristics accumulated through many years of extensive research. We
expect that this will occur also to the Jordanian putative species.
Aggression patterns
Preliminary experiments indicated that S. ehrenberg’from Jordan are polymorphic
within populations for aggression involving militants, pacifists and intermediate types
278
E. NEVO ET AL.
(i.e. high, medium and low aggression, characterizing each individual) similar to the
pattern in Israel (terms defined and polymorphism identified, as shown in Fig. 2 in
Nevo et al., 1986a). However, Jordanian females were distinctly less aggressive than
the Israeli 2n=60 females which in turn were the least aggressive among the four
Israeli species (Nevo et al., 1986a). Likewise, Egyptian Spalax from near the Sahara
desert were surprisingly nonaggressive and showed initial social evolution (Nevo et
al., 1992).Thus, the trend of decreasing aggression in steppic and desert environments
also occurred in Spalax in Jordan. It is possible that low aggression may reduce
thermal stress and death.
CONCLUSIONS
A new scenario of speciation in Jordanian Spalax
The major mechanism of speciation in the Spalacidae is Robertsonian chromosomal rearrangement, i.e. increase of chromosome numbers by fission from
biarmed to acrocentric chromosomes (Nevo, 1991; Wahrman et al., 1969a,b, 1985;
Nevo et al., 1994b, 1995),accompanied by genic divergence. The ancestral spalacid
karyotype was presumably 2n = 38 and it increased gradually, primarily by metacentric fission to 2n=62 in the Ukraine, Balkan and Anatolian ecologically harsh
steppes (Nevo, 1999; Nevo et al., 1994b, 1995). The only increase of chromosome
numbers from 2n = 60 to 2n = 62 in the Near East S. ehrenberg. superspecies were the
two individuals described here from the karyotypically polymorphic Madaba S
population (6 km south of the town), with the predominant karyotype 2n = 60. The
mechanism of their origination is yet unclear (Ivanitskaya & Nevo, 1998). We can
exclude the recent appearance of these chromosomes as a fission product due to
the G-banding patterns (Fig. 2). Chromosome no. 30 has a G-positive band in the
centromere, but its possible source, i.e. the short arm of chromosome no. 1 has a
centromeric region which is G-negative. Noteworthy, in all other animals from
Madaba S and Dhiban populations, with acrocentric condition of the first chromosome pair, chromosome no. 30 is missing. Regardless of their yet unknown
origin, however, these extra chromosomes represent an initial trend towards higher
2n, hence, a source for incipient speciation. They can become eventually fixed in
the population thereby increasing the recombination index of the cytotype which
may be better adapted to higher ecological stress towards the eastern Jordanian
desert. Pericentric inversions described both within and between species of S. ehrenbev.
in Israel (Wahrman et al., 1985) were interpreted hitherto primarily as local
chromosomal adaptatations within species rather than initiators of, or contributors
to, speciation.
The major scenario of karyotype change in Jordan is, however, not Robertsonian
and does not represent increase in 2n. All major cytotypes of S. ehrenbqi in Jordan
have 2n=60. However, the number of NFa varies geographically from 74 to 72,
68 and 70 in the Gilead, Ammon, North Moav, South Moav and Edom Mountains,
respectively. The number of biarmed chromosomes decreases (8, 7, 5 and 6 pairs)
and the number of acrocentric pairs increases, 21, 22, 24 (and even 25) and 23
pairs from Gilead to Edom Mountains, respectively. These changes that can originate
presumably by pericentric inversions and/or centromeric shifts are correlated with
SPECIATION OF MOLE RATS IN JORDAN
279
climatic diversity from semihumid to semiarid and arid climatic regimes, warm in
Moav and cooler in Edom due to the higher altitudes in the Edom Mountain range.
Thus, a clear correlation exists between climatic stress and karyotype morphology.
The latter certainly determines changes in linkage groups, and possibly also in the
recombination index (Nevo et al., 199413). However, natural selection on allozymes
through climatic stress genetically brings together several Werent karyotypes, as
was found in South Moav, but primarily in North Moav (Figs 3 and 4).
We hypothesize that speciation and adaptive radiation in the S.ehrenbe@ superspecies in Jordan utilized the scenario of initial pericentric inversion as the basis of
ecological speciation and adaptive radiation, coupled with further genic divergence,
through ecological-climatic stress. If our hypothesis is correct, we identified a new
scenario of speciation and adaptive radiation in the Spalacidae based not on
Robertsonian rearrangements,but utilizing pericentric inversions and/or centromeric
shift as a chromosomal mechanism coupled with genic divergence for the origin of
new species into increasingly stressful environments. The big Wadis (rivers and
canyons) Zarqa, Kafrayn, Mujib and Hasa provide partial or complete geographical
barriers to the newly emerging and adaptively radiating ecological species (Fig. 1).
Pencentric inversions have been described in subterranean and aboveground
rodents (King, 1993 and references therein). Hitherto pencentric inversions were
considered in the Spalacidae only in adaptations (Wahrman et al., 1969a,b, 1985)
but not in speciation. Our evidence based on ecology, genes, chromosomes and size
in Jordanian spalacids suggests for the first time the involvement of pericentric
inversions and genic differentiation in mole rat speciation (Table 3 and Fig. 4).This
hypothesis calls for future verification by multidisciplinary studies.
Pencentric inversions (PI) play a major role in chromosomal evolution, including
both adaptation and speciation in diverse groups of organisms across phylogeny
(White, 1978; King, 1993). PIS have been wrongly explained away as being
insignificant to speciation because of mechanisms avoiding malsegregation. However,
examples exist in insects, rodents and plants (see citations in King, 1993) where PIS
can produce profound hybrid infertility and/or inviability effects. Although not all
PIS cause postmating reproductive isolation, King (1993: 238-240) suggested that
mainly multiple PIS with second level effects in F2 and backcrosses are involved in
speciation. Only future studies could verify the hypothesized postmating reproductive
isolation effects of PIS in Spalax.
Here we presented karyotypic evidence and hypothesized that pericentric inversions and genic (allozymic)divergence alone may have initiated adaptive radiation
and ecological speciation in the S. ehrenbw' superspecies in Jordan, representing
speciational trends (Grant, 1989), followed by ecological adaptations through genic
(allozymic) and morphological (size) divergences to different climatic stresses. Confirmation of this hypothesis awaits the test of contact zones and potential hybridization
among the four putative species we proposed, and the demonstration of the evolution
of premating isolating mechanism, as was demonstrated in S. ehrenbw. in Israel
(Nevo, 1991).
ACKNOWLEDGEMENTS
We are deeply indebted to Mr Adnan Budieri, Head of Research and Survey,
the Royal Society for the Conservation of Nature, Jordan, for kindly permitting us
E. NEVO ETAL.
280
to collect the mole rats in the Kingdom of Jordan. We thank Avi Morgenstern and
Abdul Naser El Hueiti for field assistance. We are grateful to Carlo Redi and
Abraham Korol, and three anonymous reviewers for comments that improved the
manuscript. We thank for financial support the Israeli Ministry of Absorption; The
Human Frontiers Science Program (HFSP RG-68/95), the Israeli Discount Bank
Chair of Evolutionary Biology and the Ancell-Teicher Research Foundation for
Genetics and Molecular Evolution.
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<