Dmrt1 polymorphism and sex‐chromosome differentiation in Rana temporaria

Sex‐determination mechanisms vary both within and among populations of common frogs, opening opportunities to investigate the molecular pathways and ultimate causes shaping their evolution. We investigated the association between sex‐chromosome differentiation (as assayed from microsatellites) and polymorphism at the candidate sex‐determining gene Dmrt1 in two Alpine populations. Both populations harboured a diversity of X‐linked and Y‐linked Dmrt1 haplotypes. Some males had fixed male‐specific alleles at all markers (“differentiated” Y chromosomes), others only at Dmrt1 (“proto‐” Y chromosomes), while still others were genetically indistinguishable from females (undifferentiated X chromosomes). Besides these XX males, we also found rare XY females. The several Dmrt1 Y haplotypes differed in the probability of association with a differentiated Y chromosome, which we interpret as a result of differences in the masculinizing effects of alleles at the sex‐determining locus. From our results, the polymorphism in sex‐chromosome differentiation and its association with Dmrt1, previously inferred from Swedish populations, are not just idiosyncratic features of peripheral populations, but also characterize highly diverged populations in the central range. This implies that an apparently unstable pattern has been maintained over long evolutionary times.     

Within‐population polymorphism of sex‐determination systems in the common frog (Rana temporaria)

In sharp contrast with birds and mammals, the sex chromosomes of ectothermic vertebrates are often undifferentiated, for reasons that remain debated. A linkage map was recently published for Rana temporaria (Linnaeus, 1758) from Fennoscandia (Eastern European lineage), with a proposed sex‐determining role for linkage group 2 (LG2). We analysed linkage patterns in lowland and highland populations from Switzerland (Western European lineage), with special focus on LG2. Sibship analyses showed large differences from the Fennoscandian map in terms of recombination rates and loci order, pointing to large‐scale inversions or translocations. All linkage groups displayed extreme heterochiasmy (total map length was 12.2 cM in males, versus 869.8 cM in females). Sex determination was polymorphic within populations: a majority of families (with equal sex ratios) showed a strong correlation between offspring phenotypic sex and LG2 paternal haplotypes, whereas other families (some of which with female‐biased sex ratios) did not show any correlation. The factors determining sex in the latter could not be identified. This coexistence of several sex‐determination systems should induce frequent recombination of X and Y haplotypes, even in the absence of male recombination. Accordingly, we found no sex differences in allelic frequencies on LG2 markers among wild‐caught male and female adults, except in one high‐altitude population, where nonrecombinant Y haplotypes suggest sex to be entirely determined by LG2. Multifactorial sex determination certainly contributes to the lack of sex‐chromosome differentiation in amphibians.     

Identification of the sex‐determining region in flathead grey mullet (Mugil cephalus)

Elucidation of the sex‐determination mechanism in flathead grey mullet (Mugil cephalus) is required to exploit its economic potential by production of genetically determined monosex populations and application of hormonal treatment to parents rather than to the marketed progeny. Our objective was to construct a first‐generation linkage map of the M. cephalus in order to identify the sex‐determining region and sex‐determination system. Deep‐sequencing data of a single male was assembled and aligned to the genome of Nile tilapia (Oreochromis niloticus). A total 245 M. cephalus microsatellite markers were designed, spanning the syntenic tilapia genome assembly at intervals of 10 Mb. In the mapping family of full‐sib progeny, 156 segregating markers were used to construct a first‐generation linkage map of 24 linkage groups (LGs), corresponding to the number of chromosomes. The linkage map spanned approximately 1200 cM with an average inter‐marker distance of 10.6 cM. Markers segregating on LG9 in two independent mapping families showed nearly complete concordance with gender (R² = 0.95). The sex determining locus was fine mapped within an interval of 8.6 cM on LG9. The sex of offspring was determined only by the alleles transmitted from the father, thus indicating an XY sex‐determination system.     

Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

Isolation and Physical Mapping of Sex-Linked AFLP Markers in Nile Tilapia (Oreochromis niloticus L.)

Gynogenetically produced XX and YY Nile tilapia (Oreochromis niloticus) and diploid control groups were screened for amplified fragment length polymorphisms (AFLPs) to search for sex-linked or sex-specific markers. Family-level bulked segregant analysis (XX and YY gynogenetic family pools) and individual screening (XX and YY gynogenetics and XX and XY control individuals) identified 3 Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420) AFLP markers. OniX420 and OniY425 were shown to be allelic. Single locus polymerase chain reaction assays were developed for these markers. Tight linkage was demonstrated between the AFLP markers and the sex locus within the source families. However, these markers failed to consistently identify sex in unrelated individuals, indicating recombination between the markers and the sex-determining loci. O. niloticus bacterial artificial chromosome clones, containing the AFLP markers, hybridized to the long arm of chromosome 1. This confirmed previous evidence, based on meiotic chromosome pairing and fluorescence in situ hybridization probes obtained through chromosome microdissection, that chromosome pair 1 is the sex chromosomes.     

Geographic variation in sex‐chromosome differentiation in the common frog (Rana temporaria)

In sharp contrast with birds and mammals, sex‐determination systems in ectothermic vertebrates are often highly dynamic and sometimes multifactorial. Both environmental and genetic effects have been documented in common frogs (Rana temporaria). One genetic linkage group, mapping to the largest pair of chromosomes and harbouring the candidate sex‐determining gene Dmrt1, associates with sex in several populations throughout Europe, but association varies both within and among populations. Here, we show that sex association at this linkage group differs among populations along a 1500‐km transect across Sweden. Genetic differentiation between sexes is strongest (F_ST = 0.152) in a northern‐boreal population, where male‐specific alleles and heterozygote excesses (F_IS = ?0.418 in males, +0.025 in females) testify to a male‐heterogametic system and lack of X‐Y recombination. In the southernmost population (nemoral climate), in contrast, sexes share the same alleles at the same frequencies (F_ST = 0.007 between sexes), suggesting unrestricted recombination. Other populations show intermediate levels of sex differentiation, with males falling in two categories: some cluster with females, while others display male‐specific Y haplotypes. This polymorphism may result from differences between populations in the patterns of X‐Y recombination, co‐option of an alternative sex‐chromosome pair, or a mixed sex‐determination system where maleness is controlled either by genes or by environment depending on populations or families. We propose approaches to test among these alternative models, to disentangle the effects of climate and phylogeography on the latitudinal trend, and to sort out how this polymorphism relates to the ‘sexual races’ described in common frogs in the 1930s.     

Dmrt1 polymorphism covaries with sex‐determination patterns in Rana temporaria

Patterns of sex‐chromosome differentiation and gonadal development have been shown to vary among populations of Rana temporaria along a latitudinal transect in Sweden. Frogs from the northern‐boreal population of Ammarnäs displayed well‐differentiated X and Y haplotypes, early gonadal differentiation, and a perfect match between phenotypic and genotypic sex. In contrast, no differentiated Y haplotypes could be detected in the southern population of Tvedöra, where juveniles furthermore showed delayed gonadal differentiation. Here, we show that Dmrt1, a gene that plays a key role in sex determination and sexual development across all metazoans, displays significant sex differentiation in Tvedöra, with a Y‐specific haplotype distinct from Ammarnäs. The differential segment is not only much shorter in Tvedöra than in Ammarnäs, it is also less differentiated and associates with both delayed gonadal differentiation and imperfect match between phenotypic and genotypic sex. Whereas Tvedöra juveniles with a local Y haplotype tend to ultimately develop as males, those without it may nevertheless become functional XX males, but with strongly female‐biased progeny. Our findings suggest that the variance in patterns of sex determination documented in common frogs might result from a genetic polymorphism within a small genomic region that contains Dmrt1. They also substantiate the view that recurrent convergences of sex determination toward a limited set of chromosome pairs may result from the co‐option of small genomic regions that harbor key genes from the sex‐determination pathway.     

Identifying homomorphic sex chromosomes from wild‐caught adults with limited genomic resources

We demonstrate a genotyping‐by‐sequencing approach to identify homomorphic sex chromosomes and their homolog in a distantly related reference genome, based on noninvasive sampling of wild‐caught individuals, in the moor frog Rana arvalis. Double‐digest RADseq libraries were generated using buccal swabs from 30 males and 21 females from the same population. Search for sex‐limited markers from the unfiltered data set (411 446 RAD tags) was more successful than searches from a filtered data set (33 073 RAD tags) for markers showing sex differences in heterozygosity or in allele frequencies. Altogether, we obtained 292 putatively sex‐linked RAD loci, 98% of which point to male heterogamety. We could map 15 of them to the Xenopus tropicalis genome, all but one on chromosome pair 1, which seems regularly co‐opted for sex determination among amphibians. The most efficient mapping strategy was a three‐step hierarchical approach, where R. arvalis reads were first mapped to a low‐coverage genome of Rana temporaria (17 My divergence), then the R. temporaria scaffolds to the Nanorana parkeri genome (90 My divergence), and finally the N. parkeri scaffolds to the X. tropicalis genome (210 My). We validated our conclusions with PCR primers amplifying part of Dmrt1, a candidate sex determination gene mapping to chromosome 1: a sex‐diagnostic allele was present in all 30 males but in none of the 21 females. Our approach is likely to be productive in many situations where biological samples and/or genomic resources are limited.     

Phylogeography,more than elevation,accounts for sex chromosome differentiation in Swiss populations of the common frog (Rana temporaria)*

Sex chromosomes in vertebrates range from highly heteromorphic (as in most birds and mammals) to strictly homomorphic (as in many fishes, amphibians, and nonavian reptiles). Reasons for these contrasted evolutionary trajectories remain unclear, but species such as common frogs with polymorphism in the extent of sex chromosome differentiation may potentially deliver important clues. By investigating 92 common frog populations from a wide range of elevations throughout Switzerland, we show that sex chromosome differentiation strongly correlates with alleles at the candidate sex-determining gene Dmrt1. Y-specific Dmrt1 haplotypes cluster into two main haplogroups, Y_A and Y_B, with a phylogeographic signal that parallels mtDNA haplotypes: Y_A populations, with mostly well-differentiated sex chromosomes, occur primarily south of the main alpine ridge that bisects Switzerland, whereas Y_B populations, with mostly undifferentiated (proto-)sex chromosomes, occur north of this ridge. Elevation has only a marginal effect, opposing previous suggestions of a major role for climate on sex chromosome differentiation. The Y-haplotype effect might result from differences in the penetrance of alleles at the sex-determining locus (such that sex reversal and ensuing X-Y recombination are more frequent in Y_B populations), and/or fixation of an inversion on Y_A (as supported by the empirical observation that Y_A haplotypes might not recombine in XY_A females).     

Novel Y‐chromosome short tandem repeats in Sus scrofa and their variation in European wild boar and domestic pig populations

Y‐chromosome markers are important tools for studying male‐specific gene flow within and between populations, hybridization patterns and kinship. However, their use in non‐human mammals is often hampered by the lack of Y‐specific polymorphic markers. We identified new male‐specific short tandem repeats (STRs) in Sus scrofa using the available genome sequence. We selected four polymorphic loci (5–10 alleles per locus), falling in one duplicated and two single‐copy regions. A total of 32 haplotypes were found by screening 211 individuals from eight wild boar populations across Europe and five domestic pig populations. European wild boar were characterized by significantly higher levels of haplotype diversity compared to European domestic pigs (H_D = 0.904 ± 0.011 and H_D = 0.491 ± 0.077 respectively). Relationships among STR haplotypes were investigated by combining them with single nucleotide polymorphisms at two linked genes (AMELY and UTY) in a network analysis. A differentiation between wild and domestic populations was observed (F_ST = 0.229), with commercial breeds sharing no Y haplotype with the sampled wild boar. Similarly, a certain degree of geographic differentiation was observed across Europe, with a number of local private haplotypes and high diversity in northern populations. The described Y‐chromosome markers can be useful to track male inheritance and gene flow in wild and domestic populations, promising to provide insights into evolutionary and population genetics in Sus scrofa.     

Association of the GABRD gene and childhood‐onset mood disorders

The chromosome 1p36 region was previously indicated as a locus for susceptibility to recurrent major depressive disorder based on a linkage study in a sample of 497 sib pairs. We investigated the gamma‐aminobutyric acid A (GABA_A) δ receptor subunit gene, GABRD, as a susceptibility gene to childhood‐onset mood disorders (COMD) because of substantial evidence implicating GABAergic dysfunction in mood disorders and the position of this gene near the 1p36 linkage region. Using a sample consisting of 645 Hungarian families with a child/adolescent proband diagnosed with a mood disorder with the onset of the first episode before age 15, we found some evidence for the association of two polymorphisms located within the gene, rs2376805 and rs2376803, as well as significant evidence for biased transmission of the haplotypes of these two markers (global χ² test for haplotypes = 12.746, 3 df, P = 0.0052). Furthermore, significant evidence of association was only observed in male subjects (n = 438) when the results were analyzed by sex (χ² = 9.000 1 df, P = 0.003 for rs2376805). This was in contrast with the previous linkage findings, as LOD scores exceeding 3 were only in female–female pairs in that study. These findings point to the GABRD gene as a susceptibility gene for COMD; however, this gene may not explain the previous linkage finding.     

Genetic determination of sex and shell color in the Pacific abalone Haliotis discus hannai revealed by an integrated linkage map

Integrated linkage maps for each sex have been constructed for the Pacific abalone Haliotis discus hannai using three F₁ mapping families based on co‐dominant markers. A total of 273 markers were placed on the female map, spanning 927.3 cM with an average interval of 3.64 cM, whereas 277 markers were mapped on the male map, covering 727.0 cM with an average spacing of 2.80 cM. Both female and male maps consisted of 18 linkage groups, corresponding well with the number of chromosomes. Furthermore, the sex‐determining locus and the green/orange shell color controlling locus were mapped to the linkage group 3 (LG3) and LG9 respectively. A marker completely linked to phenotypic sex was identified, and the sex determination system was further concluded as paternal heterogametic (males XY and females XX). Based on the segregation ratio of the shell color in the progeny, a simple recessive model of epistasis was proposed to explain the distribution of different color morphs (green, orange and blue): the recessive allele determining orange type masks the effect of the locus controlling green and blue types, whereas the dominant allele at the green/orange locus permits the expression of green and blue types controlled by another locus. The current consensus map provides a useful framework for genetic studies in the Pacific abalone. Mapping of the sex‐determining locus and the shell color‐controlling locus leads to further understanding of the mechanisms underlying these important traits.     

Sex‐chromosome differentiation parallels postglacial range expansion in European tree frogs (Hyla arborea)

Occasional XY recombination is a proposed explanation for the sex‐chromosome homomorphy in European tree frogs. Numerous laboratory crosses, however, failed to detect any event of male recombination, and a detailed survey of NW‐European Hyla arborea populations identified male‐specific alleles at sex‐linked loci, pointing to the absence of XY recombination in their recent history. Here, we address this paradox in a phylogeographic framework by genotyping sex‐linked microsatellite markers in populations and sibships from the entire species range. Contrasting with postglacial populations of NW Europe, which display complete absence of XY recombination and strong sex‐chromosome differentiation, refugial populations of the southern Balkans and Adriatic coast show limited XY recombination and large overlaps in allele frequencies. Geographically and historically intermediate populations of the Pannonian Basin show intermediate patterns of XY differentiation. Even in populations where X and Y occasionally recombine, the genetic diversity of Y haplotypes is reduced below the levels expected from the fourfold drop in copy numbers. This study is the first in which X and Y haplotypes could be phased over the distribution range in a species with homomorphic sex chromosomes; it shows that XY‐recombination patterns may differ strikingly between conspecific populations, and that recombination arrest may evolve rapidly (<5000 generations).     

Sex-chromosome differentiation and ‘sex races’ in the common frog (Rana temporaria)

Sex-chromosome differentiation was recently shown to vary among common frog populations in Fennoscandia, suggesting a trend of increased differentiation with latitude. By rearing families from two contrasted populations (respectively, from northern and southern Sweden), we show this disparity to stem from differences in sex-determination mechanisms rather than in XY-recombination patterns. Offspring from the northern population display equal sex ratios at metamorphosis, with phenotypic sexes that correlate strongly with paternal LG₂ haplotypes (the sex chromosome); accordingly, Y haplotypes are markedly differentiated, with male-specific alleles and depressed diversity testifying to their smaller effective population size. In the southern population, by contrast, a majority of juveniles present ovaries at metamorphosis; only later in development do sex ratios return to equilibrium. Even at these later stages, phenotypic sexes correlate only mildly with paternal LG₂ haplotypes; accordingly, there are no recognizable Y haplotypes. These distinct patterns of gonadal development fit the concept of ‘sex races’ proposed in the 1930s, with our two populations assigned to the ‘differentiated’ and ‘semi-differentiated’ races, respectively. Our results support the suggestion that ‘sex races’ differ in the genetic versus epigenetic components of sex determination. Analysing populations from the ‘undifferentiated race’ with high-density genetic maps should help to further test this hypothesis.     

Genetic Mapping and QTL Analysis of Growth-Related Traits in the Pacific Oyster

The Pacific oyster (Crassostrea gigas) is one of the most important oysters cultured worldwide. To analyze the oyster genome and dissect growth-related traits, we constructed a sex-averaged linkage map by combining 64 genomic simple sequence repeats, 42 expressed sequence tag-derived SSRs, and 320 amplified fragment length polymorphism markers in an F₁ full-sib family. A total of 426 markers were assigned to 11 linkage groups, spanning 558.2 cM with an average interval of 1.3 cM and 94.7% of genome coverage. Segregation distortion was significant for 18.8% of the markers (P < 0.05), and distorted markers tended to occur on some genetic regions or linkage groups. Most growth-related quantitative traits were highly significantly (P < 0.01) correlated, and principal component analysis obtained four principal components. Quantitative trait locus (QTL) analysis identified three significant QTLs for two principal components, which explained 0.6–13.9% of the phenotypic variation. One QTL for sex was detected on linkage group 6, and the inheritabilities of sex for parental alleles and maternal alleles on that locus C15 are 39.8% and 0.01%, respectively. The constructed linkage map and determined QTLs can provide a tool for further genetic analysis of the traits and be potential for marker-assisted selection in C. gigas breeding.     

The red bayberry genome and genetic basis of sex determination

Morella rubra, red bayberry, is an economically important fruit tree in south China. Here, we assembled the first high‐quality genome for both a female and a male individual of red bayberry. The genome size was 313‐Mb, and 90% sequences were assembled into eight pseudo chromosome molecules, with 32 493 predicted genes. By whole‐genome comparison between the female and male and association analysis with sequences of bulked and individual DNA samples from female and male, a 59‐Kb region determining female was identified and located on distal end of pseudochromosome 8, which contains abundant transposable element and seven putative genes, four of them are related to sex floral development. This 59‐Kb female‐specific region was likely to be derived from duplication and rearrangement of paralogous genes and retained non‐recombinant in the female‐specific region. Sex‐specific molecular markers developed from candidate genes co‐segregated with sex in a genetically diverse female and male germplasm. We propose sex determination follow the ZW model of female heterogamety. The genome sequence of red bayberry provides a valuable resource for plant sex chromosome evolution and also provides important insights for molecular biology, genetics and modern breeding in Myricaceae family.     

Genetic Analysis and Molecular Mapping of a Novel Gene Conferring Resistance to Rice Stripe Virus

Rice stripe virus (RSV) is one of the most damaging diseases affecting rice in East Asia. Rice variety 502 is highly resistant to RSV, while variety 5112 is extremely susceptible. Field statistical data revealed that all “502 × 5112” F₁ individuals were resistant to RSV and the ratio of resistant to susceptible plants was 3:1 in the F₂ population and 1:1 in the BC₁F₁ population. These results indicated that a dominant gene, designated RSV1, controlled the resistance. Simple sequence repeat (SSR) analysis was subsequently carried out in an F₂ population. Sixty SSR markers evenly distributed on the 12 rice chromosomes were screened and tested. Two markers, RM229 and RM206, showed linkage with RSV1. Based on this result, six SSR markers flanking RM229 and RM206 were further selected and tested. Results indicated that SSR markers RM457 and RM473E were linked to RSV1 with a genetic distance of 4.5 and 5.0 cM, respectively. All of the four SSR markers (RM229, RM473E, RM457 and RM206) linked to RSV1 were all located on chromosome 11, therefore RSV1 should be located on chromosome 11 also. In order to find some new markers more closely linked to the RSV1 gene, sequence-related amplified polymorphism (SRAP) analysis was performed. A total of 30 SRAP primer-pairs were analyzed, and one marker SR1 showed linkage with RSV1 at a genetic distance of 2.9 cM. Finally, RSV1 gene was mapped on chromosome 11 between SSR markers RM457 and SRAP marker SR1 with a genetic distance of 4.5 cM and 2.9 cM, respectively.     

A General Model to Explain Repeated Turnovers of Sex Determination in the Salicaceae

Dioecy, the presence of separate sexes on distinct individuals, has evolved repeatedly in multiple plant lineages. However, the specific mechanisms by which sex systems evolve and their commonalities among plant species remain poorly understood. With both XY and ZW sex systems, the family Salicaceae provides a system to uncover the evolutionary forces driving sex chromosome turnovers. In this study, we performed a genome-wide association study to characterize sex determination in two Populus species, P. euphratica and P. alba. Our results reveal an XY system of sex determination on chromosome 14 of P. euphratica, and a ZW system on chromosome 19 of P. alba. We further assembled the corresponding sex-determination regions, and found that their sex chromosome turnovers may be driven by the repeated translocations of a Helitron-like transposon. During the translocation, this factor may have captured partial or intact sequences that are orthologous to a type-A cytokinin response regulator gene. Based on results from this and other recently published studies, we hypothesize that this gene may act as a master regulator of sex determination for the entire family. We propose a general model to explain how the XY and ZW sex systems in this family can be determined by the same RR gene. Our study provides new insights into the diversification of incipient sex chromosomes in flowering plants by showing how transposition and rearrangement of a single gene can control sex in both XY and ZW systems.     

Study of human SP-A,SP-B and SP-D loci: allele frequencies,linkage disequilibrium and heterozygosity in different races and ethnic groups

Background  

SP-A, SP-B, and SP-D are pulmonary surfactant proteins. Several linkage and association studies have been done using these genes as markers to locate pulmonary disease susceptibility genes, but few have studied the markers systematically in different ethnic groups. Here we studied eight markers in SP-A, SP-B, and SP-D genes in seven ethnic groups from three races (Caucasian, Black and Hispanic). We measured the similarity of the marker distribution among the ethnic groups in order to see whether people in different ethnic groups or races could be mixed together for linkage and association studies. To evaluate the usefulness of these markers, we estimated the informativeness of each marker loci in the seven ethnic groups by assessing their heterozygosity and PIC values. We also conducted linkage disequilibrium (LD) analysis to identify associated marker loci and to estimate the haplotype frequencies in each of the seven ethnic groups in an attempt to find valuable haplotypes so that the level of polymorphism of the "markers" could be increased.