Abstract
The distribution of the Fire Salamander in North Africa is discontinuous and the Edough Peninsula, Algeria, is considered as the eastern edge of the distribution area. In the current study, we establish a description of the Salamandra algira algira population in its type locality. In this context, an analysis of the mitochondrial DNA D-loop of 47 sequences comes to confirm the phylogenetic status of our population with regard to the other Algerian and Moroccan populations. Also, we used the skeletochronological method for establishing the age structure of the population. Maximum longevity reached 18 years, with a high frequency of young adults, which suggests a good survival of the juveniles. The growth of males is faster than that of the females, although the maximum size of the males is 180 mm, while that of the females is 210 mm. The Edough’s salamander’s phenotype is characterized by multiple small white spots dispersed in different parts of the body (belly, sides, legs and throat) and a high number of large red spots. These red spots are surrounded by a ring of small white spots on the lower part of the body and sometimes on the legs, thus creating specific eyespots that are often aligned along the lower sides.
Introduction
The North African region is known as a biodiversity hotspots (Mayers et al., 2000, Véla and Benhouhou, 2007; Cuttelod et al., 2008), belonging to the Mediterranean biota. Its geographical position between two barriers, the Mediterranean Sea in the North and the Sahara Desert in the South, strongly influences the distribution of biodiversity and has determined the rates of endemism in amphibians (Schleich, Kästle, and Kabisch, 1996). The Edough Massif, located in the north east of Algeria, is a good example of this biodiversity (Médail and Quézel, 1999; Véla et al., 2008), with the presence of the endemic newt Pleurodeles poireti (Gervais, 1836), together with the two other known Algerian Urodeles: the newt Pleurodeles nebulosus (Guichenot, 1850) and the salamander Salamandra algira (Bedriaga, 1883). The type-specimen of this last species was caught in this region (Eiselt, 1958). Confronted to the sub-humid and humid forests of Morocco, Algeria and Spain (Schleich et al., 1996; Donaire-Barroso and Bogaerts, 2003, Escoriza and Ben Hassine, 2014), the distribution of S. algira remains highly fragmented because suitable ecological conditions only prevail in some mountainous areas (Escoriza et al., 2006; Escoriza and Ben Hassine, 2015). However, some recent genetic studies regarding the Algerian populations of S. a. algira (Ben Hassine et al., 2016; Merabet et al., 2016) have confirmed its monophyletic origin, despite this patchy distribution. Notwithstanding, Algerian populations differ phylogenetically from the Moroccan ones, where three subspecies have been identified: S. a. tingitana, S. a. spelaea and S. a. splendens (Donaire-Barroso and Bogaerts, 2003; Escoriza and Comas, 2007; Beukema et al., 2013). The nominotypical subspecies, Salamandra algira algira appears to be endemic to Algeria (Escoriza et al., 2006; Ben Hassine et al., 2016), and the phylogenetic separation from its Moroccan sister subspecies, tingitana and splendens, dates from the Miocene. Its distribution is highly fragmented by both climatic and orographic constraints, creating “insular” mountain populations, leading to high genetic differentiation (Escoriza and Ben Hassine, 2015; Ben Hassine et al., 2016). This fragmentation toward local populations along with a west-east climatic gradient may have influenced the life-history characteristics of these populations, such as age structure, growth rate, longevity, and body size (Ento and Mastsui, 2002; Cayuela et al., 2014). Furthermore, Ben Hassine et al. (2016) suggest variations in colouration patterns between populations. Despite recent progress (Escoriza and Ben Hassine, 2015; Ben Hassine et al., 2016), the current knowledge about the ecology of S. a. algira in Algeria remains incomplete, especially with regard to population biology. As the easternmost population in the Maghreb is the most isolated one, we decided to pay particular attention to it, which generally inhabits relatively high altitudes (around 750 m) in this region that reaches an elevation of 1008 m at its highest point (Samraoui et al., 2012). In order to provide data for further comparison with other populations, our plan was threefold: 1) to specify and confirm the phylogenetic status of this population by comparing a large sample of the individual mtDNA with samples from Algeria and other regions of the Maghreb; 2) to establish body size, age structure, and growth dynamics in order to provide data concerning the profile of the population life history; 3) to accurately describe its colour phenotype to provide data for a detailed comparison with other populations.
Materials and methods
Study sites and sampling protocol
Three sites were sampled within the Edough mountain region (Fig. 1), near to Annaba (northeast of Algeria), the type locality of Salamandra algira (Eiselt, 1958). The sampling was conducted on fifty-two individuals (34 females, 14 males, and 4 juveniles) during the breeding season from October to December 2015 (Escoriza and Ben Hassine, 2014). The sampling was done during the day and after rainfall periods, through turning stones and pieces of wood. Salamanders were found in humid zones within zeen oak forests (Quercus canariensis) at the altitudes of 744 m, 762 m, and 750 m, corresponding to P1, P2, and P3, respectively. All handling was performed by the first author, immediately after capture to reduce animals’ stress. They were sexed based on their external characters (mainly cloaca shape, gravidity) (Rebelo and Leclaire, 2003). Individuals were anesthetized using ANESDERM Gé Ointment (5% lidocaïne). We measured snout-vent length (SVL) from the tip of the snout to the anterior edge of the cloaca and the tail length (TL) measured from the anterior edge of cloaca to the tip of the tail, using a calliper (0.02 mm accuracy). Salamanders were weighed using a portable Tanita-1479 scale (accuracy 0.1 g). We photographed different parts of the body for analysing colouration patterns. The third toe of one of the hind legs was clipped and then kept in Eppendorf tubes filled with 90% of ethanol. Cells of the mouth cavity were sampled using the buccal swabbing method (Pidancier, Miquel and Miaud, 2003; Poschadel and Moller, 2004). The swabs were placed in Eppendorf tubes, preserved in a glass jar with Silica gel granules to ensure good DNA conservation (Prunier et al., 2012). All the individuals survived the treatment and quickly recovered consciousness. They were then rinsed with fresh water, and released at the exact spot of their capture.
Location of the area sampled in the region of the highest summit of the Edough Massif, Annaba (North-East of Algeria). P1 site “Ain Chfa” (36°54′844″N; 7°41′085″E, 744 m altitude), P2 site “La source” (36°54′37.80″N; 7°40′04.09″E, 762 m altitude) and P3 site “Pont Romain” (36°54′14.72″N; 7°39′03.05″E, 750 m altitude).
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Location of the area sampled in the region of the highest summit of the Edough Massif, Annaba (North-East of Algeria). P1 site “Ain Chfa” (36°54′844″N; 7°41′085″E, 744 m altitude), P2 site “La source” (36°54′37.80″N; 7°40′04.09″E, 762 m altitude) and P3 site “Pont Romain” (36°54′14.72″N; 7°39′03.05″E, 750 m altitude).
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Location of the area sampled in the region of the highest summit of the Edough Massif, Annaba (North-East of Algeria). P1 site “Ain Chfa” (36°54′844″N; 7°41′085″E, 744 m altitude), P2 site “La source” (36°54′37.80″N; 7°40′04.09″E, 762 m altitude) and P3 site “Pont Romain” (36°54′14.72″N; 7°39′03.05″E, 750 m altitude).
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
DNA processing
The DNA was extracted from swabs using proteinase K (15 mg/mL) with 7% Chelex (Casquet, Thebaud and Gillespie, 2012). The three mitochondrial genes, Cytochrome b (cytb), 12S rRNA and D-loop were amplified using the following primers SalaCytbF (5′-CTAATGACCCACATCCTTCGAAAAACA-3′), SalaCytbR (3′TGGGAGTACGTATCCTACAAAGGCTG-5′) for cytb, 12SAL (5′-AAACTGGGATTAGATACCCCACTAT-3′), 16SR3 (3′-TTTCATCTTTCCCTTGCGGTAC-5′) for 12S rRNA and L-Pro-ML (5′-GGCACCCAAGGCCAAAATTCT-3′), H-12S1-ML (3′-CAAGGCCAGGACCAAACCTTTA-5′) for the D-loop fragment (Merabet et al., 2016). The final volume of PCR was 30 μl, composed as follows: 3 mL of standard buffer 10×, 0.9 μl of MgCl2 50μM, 0.6 μl of primer 10 mM (forward and reverse), 0.3 μl of BSA 100×, 0.26 μl dNTP 20 mM, 0.26 μl EUROBIOTAQ DNA pol 5 U/mL and 22.09 of purified water, with 2 μl DNA, following the same conditions and cycle numbers as in Merabet et al. (2016), whom themselves are following Steinfartz, Veith and Tautz (2000), and Beukema et al. (2010). We checked the amplification through migration of a 5-μl sample of PCR product stained with 1 μl of Blue-GelRed on 1.3% agarose gel in TAE. DNA sequencing was performed by BIOFIDAL Laboratory (Lyon, France) amidst both amplification primers.
We only used the D-loop sequences from the Algerian populations (S. algira algira) published in (Merabet et al., 2016), from Annaba (Steinfartz et al., 2000), those from the Moroccan populations (S. algira tingitana, S. algira splendens, and S. algira spelaea) were retrieved from NCBI. Clear sequence alignments were produced by eye using SEAVIEW software 4.2.12 (Gouy, Guindon and Gascuel, 2010). Cytb and 12S data were found to be highly conserved in all the sampled specimens, thus we only used D-loop sequences for further analyses. Maximum likelihood phylogenies were estimated using PhyML (Version 3.0; Guindon et al., 2010) under a GTR + G + I model of evolution. Estimation of Branch support was conducted using LRT metrics (Anisimova and Gascuel, 2006).
Age estimation
To estimate individuals age, we used the skeletochronology method (Castanet and Smirina, 1990) based on the formation of a line of arrested growth (LAGs) during periods of inactivity, especially when overwintering (Castanet, 2002). We used the phalange bones since the implied injury is mitigated by finger regeneration within one year after sampling. After removing the skin, the bone was decalcified through its immersion in a solution of nitric acid, depending on the bone diameter, at a concentration of 0.5, 1 or 3%. Cross-sections (18 and 20 μm thickness) were cut in the diaphysis region using a freezing microtome (Microm). The sections were stained with Ehrlich’s hematoxylin (Miaud, 1992; Miaud, Joly and Castanet, 1993). Sections were examined independently by at least two people using a BX51 Olympus microscope, before being photographed using a Cell-D software.
The first immediate and permanent convergence regarding the lines of arrested growth (LAGs) corresponds to the age of maturity (Fattah et al., 2014). The age of the oldest individuals can be underestimated as endosteal resorption can destroy the oldest bone layers (Miaud, 1992; Miaud et al., 2001; Castanet, 2002). In our population, we detected a clear tightening between the LAGs that usually refers to sexual maturity in salamanders. This last one is reached at three years in both sexes. As a result, we added three years if the bone section exhibited high endosteal resorption, and when the detectable LAGs were very tight, thus expressing the typical slow growth that takes place after maturity is reached. This compensation was necessary for 6 individuals. We have been aware for identifying LAGs due to the detrimental effect of drought events on growth. These LAGs are closely parallel to a LAG due to overwintyering and they are discontinuous. We used the normality test Shapiro-Wilk and Pearson correlation processed with the software R.
Colour patterns
Beside the large yellow/orange stains on the back similar to those observed in other populations, we focused on small white dots and large red spots. For each individual, we calculated the number and the location of these two kinds of marks on the different parts of the body (throat, belly, sides and legs) using photos. We regularly detected associations of the red and white dots, often combined into eyespots with small white dots encircling a larger red spot. We counted these eyespots, especially on the sides of the body where they were often aligned. The data were processed with Microsoft Excel 2010.
Results
Genetic structure of the sample
The phylogenetic tree was made up of 87 specimens’ D-loop (584 pb), including our sample of 47 individuals from the Edough Massif, the 40 samples by Merabet et al. (2016) concerning other Algerian populations, and those from GenBank for the Moroccan populations. This analysis revealed two major clades, the first group included the Moroccan subspecies Salamandra a. splendens and S. a. tingitana, while the second group included all the Algerian populations together with the Moroccan subspecies S. a. spelaea inhabiting the Beni Snassen Massif located on the eastern side of the Moulouya River (Fig. 2), hence agreeing with recent studies led by Merabet et al. (2016) and Ben Hassine et al. (2016).
Phylogenetic tree of mitochondrial DNA of Salamandra algira with the D-loop segment, using PhyML software (584 pb).
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Phylogenetic tree of mitochondrial DNA of Salamandra algira with the D-loop segment, using PhyML software (584 pb).
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Phylogenetic tree of mitochondrial DNA of Salamandra algira with the D-loop segment, using PhyML software (584 pb).
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
The Algerian samples were found to be genetically very homogeneous. However, a slight differentiation into two clades was noticeable, the first one included the Chrea sample and the second one gather together the other samples from Kabylie and the Edough Massif. The cladogram overall reflects the geography of favourable habitats. Whereas the geographic distance between our population and the other Algerian populations could explain the genetic distance, our data clearly show isolation by distance with respect to other S. algira populations of the Maghreb region for which data are available.
Biometry
In our population, female size varied from 120 to 210 mm (mean 15.5 ± 4.6 mm), while males size varied from 130 to 180 mm (mean 15.6 ± 1.03 mm). When of similar total length, females were heavier than males (WF = 0.29TLF − 28.2; WM = 0.22 TLM − 21.0). The biggest females reached 35 g while the biggest males never exceeded 19 g (Fig. 3). The correlation between size and weight was strong and positive (, , df = 33, for females and , , df = 12, for males).
Relationship between total body length (mm) and mass (g) for both females and males.
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Relationship between total body length (mm) and mass (g) for both females and males.
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Relationship between total body length (mm) and mass (g) for both females and males.
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Age structure and growth
We were able to correctly estimate the age of all the studied individuals. Bone sections revealed well-marked LAGs (Fig. 4) making it possible to recognize dark overwintering LAGs from light estivation LAGs, which remained rare. Only overwintering LAGs were used for age determination. The observed longevity was 15 years for both sexes. However, when age was corrected to compensate the loss of LAGs due to the endosteal resorption, longevity could reach 18 years. Age distribution was characterized by a large number of young individuals (the highest frequency was between 4 and 6 years), signifying high recruitment due to successful reproduction during preceding years and good juvenile survival. Age distribution also suggested a higher survival rate inside the male population than in females one (Fig. 5a).
Four examples of sections in the diaphysis of the third phalanx of the hindleg treated with Ehrlich’s hematoxylin, revealing the lines of arrested growth (LAG) formed during the wintering periods. Only well-marked lines were counted since the light ones could result from inactivity during hot summer periods (estivation). F = female; M = male; J = juvenile.
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Four examples of sections in the diaphysis of the third phalanx of the hindleg treated with Ehrlich’s hematoxylin, revealing the lines of arrested growth (LAG) formed during the wintering periods. Only well-marked lines were counted since the light ones could result from inactivity during hot summer periods (estivation). F = female; M = male; J = juvenile.
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Four examples of sections in the diaphysis of the third phalanx of the hindleg treated with Ehrlich’s hematoxylin, revealing the lines of arrested growth (LAG) formed during the wintering periods. Only well-marked lines were counted since the light ones could result from inactivity during hot summer periods (estivation). F = female; M = male; J = juvenile.
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
(A) Age structure of the Edough population of Salamandra algira. Top: females; bottom: males. (B) The relation between age and total length. Left: females; right: males.
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
(A) Age structure of the Edough population of Salamandra algira. Top: females; bottom: males. (B) The relation between age and total length. Left: females; right: males.
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
(A) Age structure of the Edough population of Salamandra algira. Top: females; bottom: males. (B) The relation between age and total length. Left: females; right: males.
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
The relationship between age and size (Fig. 5b) revealed a continuous growth throughout life (correlation , , df = 34, for females and , , df = 12, for males). Notwithstanding the fact that the sample size was not sufficient for a proper growth modeling (e.g. following von Bertalanffy’s growth equation), the shape of the curve was consistent with a logistic growth, with an inflection point at about 7 years for males and 9 years for females. Furthermore, growth dynamics varied substantially between the sexes. The males grow faster than females during the first part of adult life (on average, males reached 160-180 mm at the age of 8, whilst females reached only 140-160 mm at the same age), females maintain a faster growth rate during the second part of their adult life (after the growth inflection point). As a result, the females finally reached a bigger size than the males, with 26.5% of them exceeding 180 mm, whereas none of the sampled males reached this length (Fig. 3).
Colour patterns
Besides the usual coloration consisting of large yellow/orange spots on a dark brown/black background, most of the Edough’s salamanders also exhibited small white spots (around 1 mm), these spots are spaced out on the belly (Fig. 6B), the legs (Fig. 6A), the sides, the throat, and sometimes even over the whole body. The Edough’s salamanders also have red spots (around 2-4 mm diameter) on the paratoïd glands, the tail, and the legs. The eyespots were found on the legs or the tail and were frequently distributed according to a line (8-10 eyespots) on the sidelong of the body (Fig 6, C and D). Usually (48 individuals out of 52), these eyespots are distributed symmetrically, with the same number of eyespots (±1) on both sides. These rows of eyespots were observed on both sides of the body for 80% of males and 60% of females. Eyespots were less frequent on the legs (57.1% of males and 15.6% of females) and were never observed on the throat or on the belly (Fig. 6, E and F). The juveniles showed the same coloration as adults, white spots on different parts of the body, though eyespots were rarely observed.
Colour patterns in Salamandra algira from the Edough Massif. (A) male with red/white eyespots aligned on the lower side of the body. (B) Dorsal view of the lower limbs showing the usual yellow spots on a black/dark-brown background. White spots on the left back leg and white/red eyespots on the right back leg. (C, D) close-up shot of eyespots with a crown of large (C) and small (D) white spots encircling large red spots in two specimens. Distribution of white (E) and red (F) spots on different parts of the body in males (filled bars) and females (open bars).
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Colour patterns in Salamandra algira from the Edough Massif. (A) male with red/white eyespots aligned on the lower side of the body. (B) Dorsal view of the lower limbs showing the usual yellow spots on a black/dark-brown background. White spots on the left back leg and white/red eyespots on the right back leg. (C, D) close-up shot of eyespots with a crown of large (C) and small (D) white spots encircling large red spots in two specimens. Distribution of white (E) and red (F) spots on different parts of the body in males (filled bars) and females (open bars).
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Colour patterns in Salamandra algira from the Edough Massif. (A) male with red/white eyespots aligned on the lower side of the body. (B) Dorsal view of the lower limbs showing the usual yellow spots on a black/dark-brown background. White spots on the left back leg and white/red eyespots on the right back leg. (C, D) close-up shot of eyespots with a crown of large (C) and small (D) white spots encircling large red spots in two specimens. Distribution of white (E) and red (F) spots on different parts of the body in males (filled bars) and females (open bars).
Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003127
Discussion
This study describes an Algerian population of Salamandra algira algira in the Edough massif including samples considered as fairly representative. Our analyses confirm that this population belongs to the subspecies S. a. algira, notwithstanding the isolated character of the region thus in agreement with other recent studies (Ben Hassine et al., 2016; Merabet et al., 2016). The genetic distance from these other Algerian population was not sufficient to support the hypothesis of long-term isolation. Even the most distant sample studied (by Merabet et al., 2016), located in the Chrea Atlas Mountains, and belonging to a separate clade, was genetically not very distant from ours. Despite their great homogeneity, our samples presented a structure in two subclades that did not appear to be specific to any geographical location. Our result confirms those established by Ben Hassine et al. (2016) for the samples originating from the same region and which were classified into three groups. This could probably be an adaptation to an environmental influence or a weak interpopulation mutation rate. The S. a. algira clade is the sister group of S. a. spelaea found in the Beni-Snassen Mountains at the eastern side of the Moulouya River in Morocco (Escoriza and Comas, 2007; Beukema et al., 2010; Ben Hassine et al., 2016; Merabet et al., 2016). Our results agree with previous studies to confirm the monophyletic character of Algerian salamanders. Salamandra algira is generally found in mountainous regions and requires humid conditions and low temperatures to survive (Escoriza and Ben Hassine, 2015). The vegetation of the Edough’s Massif is dominated by Quercus suber and Quercus canariensis. These forests provide suitable conditions for salamanders. Nevertheless, further ecological studies should improve our knowledge of the specific conditions that determine the presence of S. a. algira.
In salamanders, the investment in reproduction and growth depends on rules of resources allocation that determine the dynamics of growth (Miaud, Guyetant and Faber, 2000; Miaud et al., 2001). Here, we established for the first time the age structure and growth dynamics concerning a population of S. a. algira in Algeria. The age structure and growth rates were found to be similar to that of S. algira in Morocco (Reinhard, Renner and Kupfer, 2015a, 2015b). Longevity was around 20 years for a maximum size of 210 mm. However, the skeletochronological method tends to underestimate the age, due to bone remodeling and the tightening of LAGs (Wagner et al., 2011; Fattah et al., 2014), these processes occurring mainly in populations living at high altitudes (Wagner et al., 2011; Reinhard et al., 2015a). Our population showed good growth dynamics and an age structure with a high proportion of young adults, suggesting successful reproduction during the years that preceded sampling and also a high juvenile survival. We also observed a clear difference in mass between both sexes (Fig. 3) that can be explained by the gestation of females during the activity period (Miaud et al., 2001).
Growth dynamics obeys to a logistic pattern with a slowing-down of adult growth in the middle of the adult life. This pattern is similar to the one described in S. lanzai in the French-Italian Alps (Miaud et al., 2001), with males growing faster than females during the first half of adult life, and females growing faster than males in the second half. The growth is more restricted for females during the first years of their adult life due to a greater investment in eggs and embryo development (Halliday and Verrell, 1988), yet continuous growth throughout life could provide females with a substantial fitness gain by increasing fecundity (Joly, 1991; Miaud et al., 2001; Reinhard et al., 2015b). The male investment in reproduction is generally lower than for females, and this is what leads them to reach sexual maturity earlier and at a smaller size (Caetano, Castanet and Francillon, 1985; Miaud et al., 2001). We also suspect that male size above a given threshold does not provide a significant mating advantage. A larger size could restrict their access to small refuges and lead to competition with females for large refuges and larger prey. In the future, experimental studies should confirm this hypothesis.
Compared to other Algerian populations of S. a. spelaea and S. a. algira (Escoriza and Comas, 2007; Ben Hassine et al., 2016; Merabet et al., 2016), the Edough salamanders have numerous white and red spots distributed over most of their body. They also exhibited a mixture of these two colors specifically in the form of eyespots localized on the sides and legs. These eyespots were present in higher numbers in males than in females. This could be due to the difference in size of our samples. Until the results of further studies concerning the colouration patterns in other populations, we can consider these eyespots to be frequent in the Edough population. Because of their night-time activity, we presume this white dot ring configuration improves the optical contrast between the dark background and the red spot. This could confer a badge role to these eyespots to simplify communication between individuals. To our knowledge, such a badge effect created by a red spot encircled by a lighter colour has been detected only in S. salamandra gallaica in Portugal. Behavioural studies could highlight the functional role, if any, of these eyespots.
This study provides a detailed description of the Edough population of Salamandra algira algira that can be used as a sound basis for comparison with other populations across the Maghreb. It also contributes to establishing the biological traits of Salamandra population found in one of its distribution outposts providing key elements for conservation guidelines.
Acknowledgements
We would like to thank Mohcene Allam and Amir Boulemtafes for their valuable contribution to this work. Our gratitude also goes to Khaled Merabet for his cooperation and support. All field studies were carried out with the agreement of the General Directorate of Forests, Annaba. We thank two anonymous referees for their constructive comments.
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Footnotes
Associate Editor: Matthias Stöck.